CN1681844A - HASylated polypeptides, especially HASylated erythropoietin - Google Patents

Abstract

The present invention relates to hydroxyalkyl starch (HAS) -polypeptide conjugates (HAS-polypeptides) comprising one or more HAS molecules, wherein each HAS is conjugated to the polypeptide via a carbohydrate moiety or a thioether, as well as to methods for the production of such conjugates. In a preferred embodiment, the polypeptide is Erythropoietin (EPO).

Description

HASylated polypeptides, particularly HASylated erythropoietin

The present invention relates to polypeptides, especially erythropoietin, conjugated to hydroxyalkyl starch (HSS), especially to hydroxyethyl starch.

The application of polypeptides, especially enzymes or cytokines, to the circulatory system in order to obtain a specific physiological effect is a well-known method in modern medicine.

Erythropoietin (EPO) is a glycoprotein hormone necessary for the maturation of erythroid progenitor cells into red blood cells. Adults produce EPO in the kidneys. EPO is required to regulate the level of red blood cells in the circulation. Diseases marked by low tissue oxygen levels cause increased EPO biosynthesis, which subsequently stimulates erythropoiesis. For example, in chronic renal failure, loss of renal function typically results in decreased EPO biosynthesis with concomitant erythropenia.

Erythropoietin is an acidic glycoprotein hormone with a molecular weight of approximately 34,000 Da. Human erythropoietin is a 166 amino acid polypeptide that occurs naturally as a monomer (Lin et al., 1985, PNAS 82, 7580-7584, EP 148 605 B2, EP411 678 B2). For example, US Patent 4,703,008 describes the identification, cloning and expression of the gene encoding erythropoietin. For example, US Patent 4,667,016 describes the purification of recombinant erythropoietin from cell culture medium supporting the growth of mammalian cells containing recombinant erythropoietin plasmids.

It is recognized in the art that the in vivo biological activity of EPO depends primarily on the degree to which sialic acid is bound to EPO (see, e.g., EP 428 267 B1). Theoretically, 14 sialic acid molecules could bind to one EPO molecule at the end of the carbohydrate side chains linked to N- and O-glycosylation sites. Obtaining highly sialylated EPO preparations requires very complicated purification steps.

For more detailed information on erythropoietin, see Krantz, Erythropoietin, 1991, Blood, 77(3):419-34 (Review), and Cerami, Beyond erythropoiesis: novel applications for recombinant human erythropoietin,, 2001, Semin Hematol. , (3Supp17): 33-9 (Review).

A well-known problem with peptide and enzyme applications is that these proteins often do not exhibit satisfactory stability. In particular, erythropoietin has a relatively short plasma half-life (Spivak and Hogans, 1989, Blood 73, 90; McMahon et al., 1990, Blood 76, 1718). This means that therapeutic plasma levels decrease rapidly, necessitating repeated intravenous administration. Furthermore, in some cases, an immune response against these peptides was also observed.

It is generally believed that when polypeptides are conjugated to polymeric molecules, the stability of the polypeptides can be increased and the immune response against these polypeptides reduced. WO 94/28024 discloses that physiologically active polypeptides modified with polyethylene glycol (PEG) show reduced immunogenicity and antigenicity, and their circulation in the blood stream is much longer than that of unconjugated proteins, that is, the time required for clearance is longer.

However, PEG-drug conjugates also have several disadvantages, for example, they do not have a native structure that can be recognized by components of degradation pathways in vivo. Therefore, in addition to PEG-conjugates, other conjugates and protein polymers were prepared. Various methods of crosslinking different proteins and macromolecules such as polymerases have been described in the literature (see, for example, Wong, Chemistry of protein conjugation and cross-linking, 1993 , CRCS, Inc.).

Hydroxyethyl starch (HES) is a naturally occurring amylopectin derivative that is degraded by alpha-amylases in vivo. The preparation of HES-protein conjugates is known in the art (see, for example, HES-hemoglobin conjugates in DE 26 16 086 or DE 26 46 854).

DE 26 46 854 discloses a method for coupling hemoglobin to HES. In these methods, HES is reacted with sodium periodate to produce a dialdehyde that is linked to hemoglobin. Different from this, in the coupling method of hemoglobin and HES disclosed in DE 2616 086, a cross-linking agent (such as bromocyane) is first combined with HES, and then hemoglobin is connected with an intermediate product.

HES is a substituted derivative of the carbohydrate polymer pullulan, present in concentrations up to 95% by weight in corn starch. HES shows favorable biological properties and can be used as a blood volume replacement agent in clinical hemodilution therapy (Sommermeyer et al., 1987, Krankenhauspharmazie, 8(8), 271-278; Weidler et al., 1991, Arzneim.- Forschung/Drug Res., 41, 494-498).

Amylopectin consists of a glucose moiety in which α-1,4-glycosidic linkages are present in the main chain and α-1,6-glycosidic linkages at branch sites. The physicochemical properties of this molecule mainly depend on the type of glycosidic linkage. Since the α-1,4-glycosidic bond has a cut, a helical structure with about 6 glucose monomers per turn is produced.

The physicochemical as well as biochemical properties of the polymer can be altered by substitution. Hydroxyethyl groups can be introduced by base hydroxyethylation. By varying the reaction conditions, it was possible to study the different reactivity of each hydroxyl group in the unsubstituted glucose monomer to hydroxyethylation. Due to this fact, the skilled person is able to influence the substitution pattern to a limited extent.

Therefore, HES is mainly characterized by molecular weight distribution and degree of substitution. There are two ways to describe the degree of substitution:

1. The degree of substitution (DS) is described as the ratio of substituted glucose monomers to all glucose moieties.

2. The degree of substitution is described in terms of "molar substitution" (MS), which describes the number of hydroxyethyl groups per glucose moiety.

HES solutions exist as polydisperse compositions, with each molecule differing from each other in terms of degree of polymerization, number and pattern of branching sites, and substitution pattern. HES is therefore a mixture of compounds of different molecular weights. Therefore, a specific HES solution was determined according to the average molecular weight using a statistical method. Herein, M _n is calculated as an arithmetic mean depending on the number of molecules. Furthermore, the weight average _Mw represents a unit depending on the mass of the HES.

The HES-drug conjugates disclosed in the art have the disadvantage that HES cannot be site-specifically conjugated to a drug. Thus, this coupling produces very heterogeneous products containing multiple components that may not be active due to the disruption of the three-dimensional structure during the coupling step.

In conclusion, there remains a need for further improved polypeptides with increased stability and/or biological activity. This is especially true for erythropoietin, isoforms with high sialic acid content and therefore high activity must be purified from isoforms with low sialic acid content (see EP 428 267 B1). Therefore, it would be highly advantageous if production methods could be employed that would yield highly active polypeptides without the need for complex purification. Unfortunately, producing polypeptides in bacterial or insect cells is often difficult because often the resulting polypeptides are not properly folded and in their native conformation, and lack proper glycosylation.

Therefore, an object of the present invention is to provide polypeptide derivatives, especially erythropoietin derivatives, which have high biological activity in vivo and can be easily produced at low cost. Furthermore, another object of the present invention is to provide a method for producing polypeptide derivatives, which is easy to perform and produces products with high biological activity. Another object of the present invention is to provide pharmaceutical compositions containing polypeptide derivatives with high biological activity.

According to one aspect of the invention, this problem is solved with a hydroxyalkyl starch (HSS)-erythropoietin (EPO)-conjugate (HAS-EPO) containing one or more HAS molecules, wherein each HAS is associated with EPO passed

a) the carbohydrate portion; or

b) Thioether coupling.

The HAS-EPO of the present invention has the following advantages: Compared with the erythropoietin before coupling, the biological stability is improved. In addition, it also exhibits higher biological activity than standard BRP EPO. This is mainly due to the fact that HAS-EPO is rarely or even not recognized by the clearance systems of the liver and kidneys and therefore persists in the circulation for a long time. In addition, since HAS is linked in a site-specific manner, the risk of disruption of the in vivo biological activity of EPO due to the coupling of HAS to EPO is minimized.

The HAS-EPO of the present invention mainly contains two components, ie erythropoietin (EPO) polypeptide and hydroxyalkyl starch (HSS) linked thereto.

EPO can be of any human origin (see, e.g., Inoue, Wada, Takeuchi, 1994, An improved method for the purification of humanerythropoietin with high in vivo activity from the urine of anemic patients, Biol Pharm Bull. 17(2), 180- 4; Miyake, Kung, Goldwasser, 1977, Purification of human erythropoietin, J BiolChem., 252 (15), 5558-64) or another mammalian source, which can be obtained from naturally occurring sources (such as human kidney, human embryo Liver, or animal (preferably monkey kidney) obtained by purification. Furthermore, the expression "erythropoietin" or "EPO" also includes EPO variants with erythropoietic activity, in which one or more amino acids (for example 1-25, preferably 1-10, more preferably 1-5, Most preferably 1 or 2) have been substituted by further amino acids (see, e.g. EP 640 619 B1). Measurement of erythropoietic activity is described in the art (for measurements of in vitro activity see e.g. Fibi et al., 1991, Blood, 77, 1203 ff; Kitamura et al., 1989, J. Cell Phys., 140, 323-334; See Ph.Eur.2001,911-917 about the measurement of EPO in vivo activity; Ph.Eur.2000,1316Erythropoietini solutio concentraterata, 780-785; European Pharmacopoeia (1996/2000); 8, 371-377; Fibi, Hermentin, Pauly, Lauffer, Zettlmeissl., 1995, N-and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells, Blood, 85(5), 1229-36; (in On days 1, 2, and 3, female NMRI mice were injected with EPO and modified EPO (equivalent protein 50 ng/mouse), and blood samples were collected on day 4 to measure reticulocytes)). Other publications measuring EPO activity assays include Barbone, Aparicio, Anderson, Natarajan, Ritchie, 1994, Reticulocytes measurements as a bioassay for erythropoeitin, J.Pharm.Biomed.Anal., 12(4), 515-22; Bowen, Culligan, Beguin, Kendall, Vlllis, 1994, Estimation of effective and total erythropoiesis inmyelodysplasia using serum transferring receptor and erythropoietin concentrations, with automated reticulocyte parameters, Leukemi, 8(1), 151-5; , 1992, Role of glycosylation on the secretion and biological activity of erythropoietin, Biochemistry, 31(41), 9871-6; Higuchi, Oh-eda, Kuboniwa, Tomonoh, Shimonaka, Ochi, 1992; Role of sugar chains in the expression of the biology activity of human erythropoietin, J.Biol.Chem., 267(11), 7703-9; Yamaguchi, Akai, Kawanishi, Ueda, Masuda, Sasaki, 1991, Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties, J.Biol.Chem., 266(30), 20434-9; Takeuchi, Inoue, Strickland, Kubota, Wada, Shimizu, Hoshi, Kozutsumi, Takasaki, Kobata, 1989, Relationship between sugar chain structure and biological activity of recombinanthuman erythropoietin produced in Chinese hamster ovary cells, Proc.Natl.Acad.Sci.USA, 85(20), 7819-22; Kurtz, Eckardt, 1989, Assay methods for erythropoietin, Nephron., 51(1), 11-4 (German); Zucali, Sulkowski, 1985, Purification of humanurinary erythropoietin on controlled-pore glass and silicicacid, Exp.Hematol., 13(3), 833-7; Krystal, 1983, Physical and biological characterization of erythroblast enhancing factor), (EEF a late acting erythropoeticstimulator in serum distinct from erythropoietin, Exp. Hematol., 11(1), 18-31.

Preferably, EPO is produced recombinantly. This includes production in eukaryotic or prokaryotic cells, preferably mammalian, insect, yeast, bacterial cells or any other cell type suitable for recombinant production of EPO. Furthermore, EPO can also be expressed in transgenic animals (for example in body fluids such as milk, blood, etc.), in transgenic birds, especially poultry, preferably chicken eggs, or in transgenic plants.

Recombinant production of polypeptides is well known in the art. It usually involves transfecting host cells with an appropriate expression vector, culturing the host cells under conditions capable of producing the polypeptide, and purifying the polypeptide from the host cells. For details see e.g. Krystal, Pankratz, Farber, Smart, 1986, Purification of human erythropoietin tohomogeneity by a rapid five-step procedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant humanerythropoietin produced using a baculovirus vector, Blood, 74(2), 652-7; EP 640 619 B1 and EP 668 351 B1.

In a preferred embodiment, the EPO has the amino acid sequence of human EPO (see EP 148605 B2).

EPO may contain one or more carbohydrate side chains (preferably 1-4, preferably 4) attached to EPO by N- and/or O-linked glycosylation, ie EPO is glycosylated. When EPO is produced in eukaryotic cells, the polypeptide is usually post-translationally glycosylated. Thus, carbohydrate side chains can be attached to EPO during biosynthesis in mammalian, especially human, insect or yeast cells. The structure and properties of glycosylated EPO have been extensively studied in this field (see EP 428 267 B1; EP 640 619 B1; Rush, Derby, Smith, Merry, Rogers, Rohde, Katta, 1995, Microheterogeneity of erythropoietin carbohydrate structure, Anal Chem ., 67(8), 1442-52; Takeuchi, Kobata, 1991, Structures and functional roles of the sugar chains of human erythropoietins, Glycobiology, 1(4), 337-4 6 (Review).

HAS can be directly coupled to EPO, or it can be coupled through a linker molecule. The nature of the linker molecule depends on the way HAS is linked to EPO. Table 1 and below describe possible linker functional groups. Several linkers are commercially available (eg, from Pierce, available from Perbio Science Deutschland GmbH, Bonn, Germany)). Table 2 describes some suitable linkers. The nature of the linker and its use are described in detail below in the section on HES-EPO production methods.

According to a preferred embodiment of the HAS-EPO conjugate of the invention, HAS is coupled to EPO via a carbohydrate moiety.

In the context of the present invention, the term "carbohydrate moiety" refers to hydroxyaldehydes or hydroxyketones, as well as their chemical modifications (see Römpp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9th Edition 1990, Volume 9, 2281-2285, and here references cited there). In addition it refers to derivatives of naturally occurring carbohydrate moieties such as glucose, galactose, mannose, sialic acid, and the like. The term also includes naturally occurring carbohydrate moieties whose ring structures have been opened and chemically oxidized.

Carbohydrate moieties can be directly linked to the EPO polypeptide backbone. Preferably, the carbohydrate moiety is part of a carbohydrate side chain. In such cases, there may also be other carbohydrate moieties between the HAS-linked carbohydrate moiety and the backbone of the EPO polypeptide. More preferably, the carbohydrate moiety is the terminal portion of a carbohydrate side chain.

In a more preferred embodiment, the HAS is coupled to the galactose residue of the carbohydrate side chain, preferably to the terminal galactose residue of the carbohydrate side chain. This galactose residue can be used for conjugation by removal of the terminal sialic acid followed by oxidation (see below).

In another more preferred embodiment, the HAS is coupled to the sialic acid residue of the carbohydrate side chain, preferably to the terminal sialic acid residue of the carbohydrate side chain.

In addition, HAS can also be coupled with EPO via thioether. As detailed below, the S atom can be derived from any naturally or non-naturally occurring SH group attached to EPO.

In a preferred embodiment, the S atom may be derived from a SH group (see below) introduced into the oxidized carbohydrate moiety of HES, preferably the oxidized carbohydrate moiety as part of the carbohydrate side chain of EPO.

Preferably, the S atom in the thioether is derived from naturally occurring cysteine or added cysteine. More preferably the EPO has the amino acid sequence of human EPO, the naturally occurring cysteines being cysteine 29 and/or 33. In a more preferred embodiment, HAS is coupled to cysteine 29 and cysteine 33 is replaced by another amino acid. In addition, HAS can also be conjugated to cysteine 33, while cysteine 29 is replaced by another amino acid.

In the present invention, the term "added cysteine" means that the polypeptide, preferably EPO, contains a cysteine residue that is not present in the wild-type polypeptide.

In this aspect of the invention, cysteine may be an additional amino acid added at the N- or C-terminus of EPO.

In addition, cysteine can also be added by substituting naturally occurring amino acids for cysteine. Suitable methods are known in the art (see above). In the context of this aspect of the invention, the EPO is preferably human EPO and the substituted amino acid residue is preferably serine126.

The second component of HAS-EPO is hydroxyalkyl starch (HAS).

In the present invention, the term "hydroxyalkyl starch" refers to starch derivatives substituted with hydroxyalkyl groups. Here, the alkyl group may be substituted. Preferably, the hydroxyalkyl group contains 2-10 carbon atoms, more preferably 2-4 carbon atoms. Therefore, "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are preferred.

The hydroxyalkyl group of HAS contains at least one OH group.

The expression "hydroxyalkylstarch" also includes derivatives in which the alkyl groups are mono- or polysubstituted. In this regard, it is preferred that the alkyl group is substituted by halogen, especially fluorine, or by aryl, as long as the HAS remains water-soluble. In addition, the terminal hydroxyl group of the hydroxyalkyl group may also be esterified or etherified. In addition, the alkyl group of hydroxyalkyl starch can also be linear or branched.

In addition, linear or branched substituted or unsubstituted alkenyl groups can also be used instead of alkyl groups.

For all embodiments of the invention, hydroxyethyl starch (HES) is most preferred.

In the present invention, the average molecular weight (weight average) of hydroxyethyl starch may be 1-300 kDa, wherein the average molecular weight of 5-100 kDa is more preferred. The hydroxyethyl starch may further exhibit a molar substitution degree of 0.1-0.8 relative to the hydroxyethyl group and a C2:C6 substitution ratio of 2-20.

Each EPO molecule of HAS-EPO may contain 1-12, preferably 1-9, 1-6 or 1-3, most preferably 1-4 HAS molecules. The number of HAS molecules per EPO molecule can be determined by quantitative carbohydrate composition analysis using GC-MS after hydrolysis of the product and derivatization of the resulting monosaccharides (see Chaplin and Kennedy (eds.), 1986, "Carbohydrate Analysis: A "Carbohydrate Analysis: apractical approach", IRL Press Practical approach series (ISBN 0-947946-44-3), for details, see Chapter 1, Monosaccharides, pages 1-36; Chapter 2, Oligosaccharides, pp. 37-53, Chapter 3, Neutral Polysaccharides, pp. 55-96).

The HAS-EPO conjugate of the present invention can exhibit substantially the same in vitro biological activity as the recombinant natural EPO, because the in vitro biological activity only depends on the binding affinity of the EPO receptor. Methods for assaying in vitro biological activity are well known in the art (see above).

In addition, HAS-EPO also showed higher in vivo activity than EPO used as the starting material for conjugation (unconjugated EPO). Methods for assaying biological activity in vivo are well known in the art (see above). In addition, Examples 9 and 10 also give assays for measuring EPO activity in vivo and in vitro.

If the in vivo activity of unconjugated EPO is taken as 100%, the HAS-EPO conjugate may show 110-500%, preferably 300-400%, or 110%-300%, preferably, 110%-200% , more preferably 110%-180%, or 110-150%, most preferably 110%-140% in vivo activity.

Compared with Amgen's highly sialylated EPO (see EP 428 267 B1), if the in vivo activity of highly sialylated EPO is set to 100%, HAS-EPO shows preferably at least 50%, more preferably at least 70%, more preferably at least 85%, or at least 95%, at least 150%, at least 200%, or at least 300% in vivo activity of highly sialylated EPO. Most preferably, it exhibits at least 95% of the in vivo activity of highly sialylated EPO.

The high in vivo bioactivity of the HAS-EPO conjugates of the present invention is mainly based on the fact that the HAS-EPO conjugates remain in circulation longer than unconjugated EPO because it is less recognized by the liver clearance system , and decreased renal clearance due to higher molecular weight. The method of measuring EPO circulating half-life in vivo is known in the art (Sytkowski, Lunn, Davis, Feldman, Siekman, 1998, Human erythropoietin dimmers with markedly enhanced in vivo activity, Proc.Natl.Acad.Sci.USA, 95 (3), 1184 -8).

It is therefore a distinct advantage of the present invention to provide HAS-EPO which can be administered less frequently than EPO preparations currently available commercially. Standard EPO preparations must be administered for at least 3 consecutive days, whereas the HAS-EPO conjugate of the present invention is preferably administered twice a week, more preferably once a week.

All the embodiments disclosed below with respect to the method for producing HAS-EPO according to the invention relating to the properties of EPO or HAS also apply to the HAS-EPO conjugates according to the invention.

Hydroxyalkyl starch is an ether derivative of starch. In addition to the ether derivatives mentioned, other starch derivatives can also be used in the present invention. For example, derivatives containing esterified hydroxyl groups may be used. These derivatives may be, for example, derivatives of unsubstituted monocarboxylic or dicarboxylic acids having 2 to 12 carbon atoms, or derivatives of substituted derivatives thereof. Particularly useful are derivatives of unsubstituted monocarboxylic acids having 2 to 6 carbon atoms, especially derivatives of acetic acid. For this purpose, acetyl starch, butyl starch or propyl starch are preferred.

In addition, derivatives of unsubstituted dicarboxylic acids containing 2 to 6 carbon atoms are also preferred.

For derivatives of dicarboxylic acids, it is advantageous that the second carboxyl group of the dicarboxylic acid is also esterified. In addition, monoalkyl ester derivatives of dicarboxylic acids are also suitable in the present invention.

For substituted monocarboxylic or dicarboxylic acids, the substituents may preferably be the same as those described above for substituting alkyl residues.

The technique of esterification of starch is known in the art (see, for example, Klemm D. et al., "Comprehensive Cellulose Chemistry" (Comprehensive Cellulose Chemistry) Vol. 2, 1998, Whiley-VCH, Weinheim, New York, see in particular Chapter 4.4, Esterification of Cellulose (ISBN 3-527-29489-9).

In another aspect, the present invention relates to a method for producing a hydroxyalkyl starch (HAS)-erythropoietin (EPO) conjugate (HAS-EPO), comprising the steps of:

a) providing EPO capable of reacting with the modified HAS,

b) providing a modified HAS capable of reacting with EPO in step a), and

c) reacting the EPO of step a) with the HAS of step b), thereby producing a HAS-EPO containing one or more HAS molecules, wherein each HAS passes through the EPO

i) the carbohydrate portion; or

ii) Thioether coupling.

The method of the present invention has the advantage of producing HAS-EPO conjugates exhibiting high biological activity. In addition, the method of the present invention also has the advantage of being able to produce potent EPO derivatives at low cost, because the method does not include complex and time-consuming purification steps leading to low final yields, for example, it does not need to purify away In vivo bioactive or hyposialylated EPO forms without in vivo bioactivity. In particular, Example 20 demonstrates that a HES-EPO produced with minimal modification steps exhibited 3-fold the activity of standard BRP-EPO.

Thus, in the first step of the method of the invention, an EPO capable of reacting with the modified HAS is provided.

The term "provided" as used in the present invention is interpreted that after each step a molecule with the desired properties (EPO in step a) and HAS in step b) can be obtained.

For step a) this involves purification of EPO from natural sources, as well as recombinant production in host cells or organisms and, if necessary, modification of the EPO thus obtained.

Everything that has been said about EPO as the starting material of the invention applies equally to erythropoietin as part of the HAS-EPO conjugate of the invention. In this regard, the preferred embodiments disclosed above also apply to the method according to the invention.

Therefore, in a preferred embodiment, EPO has the amino acid sequence of human EPO.

EPO is preferably produced recombinantly. Production in eukaryotic or prokaryotic cells is included, preferably in mammalian, insect, yeast, bacterial cells or in any other cell type suitable for recombinant production of EPO. Furthermore, EPO can also be expressed in transgenic animals (for example in body fluids such as milk, blood, etc.), in transgenic birds, especially poultry, preferably chicken eggs, or in transgenic plants.

Recombinant production of polypeptides is well known in the art. It usually includes transfecting host cells with an appropriate expression vector, culturing host cells under conditions capable of producing polypeptides, and purifying polypeptides from host cells (Krystal, Pankratz, Farber, Smart, 1986, Purification of human erythropoietin to homogeneity by a rapidfive-step procedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant human erythropoietin producing a baculoviru vector, Blood, 74(2), 652 -7; EP 640 619 B1 and EP 668 351 B1).

EPO may contain one or more carbohydrate side chains attached to EPO by N- and/or O-linked glycosylation, ie, EPO is glycosylated. When EPO is produced in eukaryotic cells, the polypeptide is usually post-translationally glycosylated. Thus, carbohydrate side chains can be attached to EPO during biosynthesis in mammalian, especially human, insect or yeast cells, either from transgenic animals (see above), or derived from of or still remain in the animal.

These carbohydrate side chains can be chemically or enzymatically modified after expression in an appropriate cell, for example by removing or adding one or more carbohydrate moieties (see, e.g., Dittmar, Conradt, Hauser, Hofer, Lindenmaier, 1989, Advances in Protein Design (Advances in Protein design); Edited by Bloecker, Collins, Schmidt and Schomburg, GBF-Monographs 12, 231-246, VCH Publishers, Weinheim, New York, Cambridge).

The object of the method of the present invention is to provide a HAS-EPO comprising one or more HAS molecules, wherein the HAS is coupled to the EPO via a carbohydrate moiety (i) or via a thioether (ii). Accordingly, the EPO provided by step a) should have the property of being able to be coupled via carbohydrate moieties and/or via thioethers. Therefore, after step a), EPO may preferably contain

(1) at least one reactive group attached directly or via a linker molecule to a sulfhydryl group or a carbohydrate moiety, capable of reacting with HES or modified HES,

(2) a carbohydrate moiety to which at least one modified HAS can be coupled, and/or

(3) At least one free SH group.

For possibility (1) above, the EPO of step a) can preferably be obtained by coupling appropriate linker molecules to SH groups or carbohydrate moieties of EPO. 2.1 in Example 4 provides an example of such a modified EPO. It is important to ensure that the addition of adapter molecules does not damage EPO. And this is well known to those skilled in the art.

For possibility (2) above, in a preferred embodiment, the modified HAS is coupled to EPO via a carbohydrate moiety.

Carbohydrate moieties can be directly linked to the EPO polypeptide backbone. Preferably, the carbohydrate moiety is part of a carbohydrate side chain. In this case, there may also be other carbohydrate moieties between the carbohydrate moiety attached to the HAS and the backbone of the EPO polypeptide. More preferably, the carbohydrate moiety is the terminal portion of a carbohydrate side chain.

Thus, in a preferred embodiment, the modified HAS is linked (via a linker or no linker, see below) to a carbohydrate chain which is linked to the N- and/or O-glycosylation site of EPO.

However, it is also contemplated by the invention that the EPO contains other carbohydrate moieties coupled to the modified HAS. Techniques for linking carbohydrate moieties to polypeptides enzymatically or by genetic engineering, and subsequent expression in appropriate cells are known in the art (Berger, Greber, Mosbach, 1986, Galactosyltransferase-dependent sialylation of complex and endo-Nacetylglucosaminidase H-treated coreN-glycans in vitro, FEBS Lett., 203(1), 64-8; Dittmar, Conradt, Hauser, Hofer, Lindenmaier, 1989, Advances in Protein Design; edited by Bloecker, Collins, Schmidt and Schomburg, GBF-Monograph, 12, 231 -246, VCHPubishers, Weinheim, New York, Cambridge).

In a preferred embodiment of the method according to the invention, the carbohydrate moiety is oxidized in order to be able to react with the modified HAS. This oxidation can be carried out chemically or enzymatically.

Methods for chemically oxidizing carbohydrate moieties of polypeptides are well known in the art and include treatment with periodate (Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).

By chemical oxidation it is in principle possible to oxidize any carbohydrate moiety, terminal or not. However, by choosing mild conditions (1 mM periodate, 0 °C, as opposed to stringent conditions: 10 mM periodate, 1 h at room temperature), it is possible to preferentially oxidize terminal carbohydrate moieties of carbohydrate side chains, such as sialic acid or galactose.

In addition, carbohydrate moieties can also be oxidized enzymatically. Enzymes for oxidizing individual carbohydrate moieties are well known in the art, eg for galactose the enzyme is galactose oxidase.

If the terminal galactose moiety is to be oxidized, if EPO is produced in cells capable of linking sialic acid to carbohydrate chains, e.g. complete) removal of terminal sialic acid. Chemical or enzymatic methods for removing sialic acid are well known in the art (Chaplin and Kennedy (eds.), 1996, "Carbohydrate Analysis: a practical approach", see in particular Chapter 5, Montreuill, Sugar Proteins, pp. 175-177; IRL Press Practical approach series (ISBN 0-94794 6-4 4-3)).

The invention also includes, in step a) linking the carbohydrate moiety to be linked to the modified HAS to EPO. If it is desired to attach galactose, this can be achieved using galactosyltransferase. These methods are well known in the art (Berger, Greber, Mosbach, 1986, Galactosyltransferase-dependent sialylation of complex andendo-N-acetylglucoaminidase H-treated core N-glycans in vitro, FEBS Lett., 203(1), 64-8) .

In a most preferred embodiment, in step a), if necessary, preferably after partial or complete removal (enzymatically and/or chemically) of the terminal sialic acid, by oxidation of at least one of the one or more carbohydrate side chains of EPO EPO is modified with a terminal sugar unit (preferably galactose) (see above).

Thus, preferably, the modified HAS is coupled to the oxidized terminal sugar unit of the carbohydrate chain, preferably to galactose.

Furthermore, the modified HAS can preferably also be coupled to a terminal sialic acid, which is preferably oxidized in step a) of the process according to the invention.

In a further preferred embodiment (see point (3) above), EPO contains at least one free SH group.

According to a preferred embodiment, the SH group can be attached to a preferably oxidized carbohydrate moiety, for example by using hydroxylamine derivatives, such as 2-(aminooxy)ethanethiol hydrochloride (Bauer L. et al., 1965, J.Org.Chem., 30, 949), or by using hydrazide derivatives, such as thioglycolic acid hydrazide (Whitesides et al., 1977, J.Org.Chem., 42, 332). The method of coupling these molecules to the oxidized carbohydrate moiety of EPO can be similar to that described in Example Schemes 8 and 9.

In a further preferred embodiment, the free SH group is part of a naturally occurring cysteine or an added cysteine.

Mammalian EPO contains several cysteines that normally form disulfide bonds. However, EPO with at least one naturally occurring cysteine containing a free SH group can be obtained by substituting at least one cysteine for another amino acid, for example by recombinant means. Amino acid substitution methods are well known in the art (Elliott, Lorenzini, Chang, Barzilay, Delorme, 1997, Mapping of the active site of recombinant humanerythropoietin, Blood, 89(2), 493-502; Boissel, Lee, Presnell, Cohen, Bunn , 1993, Erythropoietin structure-function relationships. Mutant proteins that test a model of tertiary structure, J Biol Chem., 268(21), 15983-93)).

Preferably, EPO has the amino acid sequence of human EPO, and the naturally occurring cysteines are cysteines 29 and/or 33.

Thus, in a preferred embodiment, cysteine 33 is replaced by another amino acid and in step c) the modified HAS is coupled to cysteine 29.

In a further preferred embodiment, cysteine 29 is replaced by another amino acid and in step c) the modified HAS is coupled to cysteine 33.

In the present invention, the term "added cysteine" means that the polypeptide (preferably EPO) contains a cysteine residue which is not present in the wild-type polypeptide. This can be accomplished by adding (eg, by recombinant means) a cysteine residue at the N- or C-terminus of the polypeptide, or by replacing (eg, by recombinant means) a naturally occurring amino acid with cysteine. These methods are well known to those skilled in the art (see above).

Preferably, cysteine is added by substituting a naturally occurring amino acid for a cysteine.

In a preferred embodiment, the EPO is human EPO and the amino acid residue to be substituted is Serine126.

Preferably, the modified HAS is coupled with the added cysteine in step c).

In step b) of the method of the invention, a modified HAS capable of reacting with the EPO of step a) is provided.

For this purpose, HAS may preferably be modified at its reducing end. This has the advantage that the chemical reaction is easy to control and the skilled person can be sure which group of HAS is modified during the reaction. Since only one group is introduced into HAS, cross-linking of different EPO molecules through multifunctional HAS molecules and other side reactions can be prevented.

Therefore, the modified HAS is able to react with the following components:

(1) at least one group connected directly or via a linker molecule to a sulfhydryl group or a carbohydrate moiety of EPO,

(2) at least one carbohydrate moiety, preferably it is oxidized, and/or

(3) At least one free SH group.

Regarding point (1) above, the modification of HAS depends on the group attached to EPO. The mechanism is well known in the art. 2.1 in Example 4 gives an example.

Regarding the above points (2) and (3), several methods of modifying HAS are known in the art. The rationale for these approaches is either to modify the reactive groups of HAS to be able to react with carbohydrate moieties or SH groups, or to couple a linker molecule to HAS containing reactive groups capable of reacting with carbohydrate moieties or SH groups. group.

For point (2), the modified HAS is capable of reacting with oxidized carbohydrate moieties, preferably with terminal sugar residues, more preferably with galactose or with terminal sialic acid.

Several methods are known to modify HAS so that it can react with oxidized preferred terminal sugar residues. As mentioned above, this modification can be introduced selectively in the reducing end region of the HES-chain. In this case, in the first step, the aldehyde group is oxidized to the lactone. These modifications include, but are not limited to, the addition of hydrazide, amino (and hydroxylamino), semicarbazide, or thiol functional groups to HAS, either directly or through a linker. These techniques are described in further detail in Examples 2-4. Furthermore, the mechanism itself is well known in the art (see, e.g. DE19628705A1; Hpoe et al., 1981, Carbohydrate Res., 91, 39; Fissekis et al., 1960, Journal of Medicinal and Pharmaceutical Chemistry, 2, 47; Frie, 1998, Thesis, Fachhochschule Hamburg, DE).

In the present invention, the addition of hydrazide or hydroxylamino functional groups is preferred. In this case, the selective reaction of the modified HAS with the oxidized carbohydrate moieties of EPO is ensured, preferably without lysine side chains with oxidized sugars, by carrying out the reaction of step c) of the process according to the invention at pH 5.5 The residues form imines to cause intermolecular or intramolecular EPO crosslinking.

Regarding point (3), several methods are known for modifying HAS so that it can react with free SH groups. Preferably, this modification is introduced selectively in the reducing end region of the HES-chain. These methods include, but are not limited to, the addition of maleimide, disulfide, or haloacetamide functional groups to HAS. These techniques are described in further detail in Examples 2-4.

For further details on these techniques, see Chamov et al., 1992, J.Biol.Chem., 267, 15916; Thorpe et al., 1984, Eur.J.Biochem., 140, 63; Greenfield et al., 1990, Cancer Research, 50, 6600, and references cited in 1.3 of Example 2.

Table 1 lists some other possible functional groups, providing a systematic overview of possible linker molecules. Furthermore, the mechanisms themselves are well known in the art.

Several linker molecules that can be used in the present invention are known in the art or are commercially available (eg from Pierce, available from Perbio Science Deutschland GmbH, Bonn, Germany). Table 2 lists examples.

In step c) of the process of the invention, the EPO of step a) is reacted with the HAS of step b) to produce HAS-EPO containing one or more HAS molecules, wherein HAS is coupled to EPO via a carbohydrate moiety or via a thioether couplet.

In principle, the detailed method for reacting EPO with modified HAS depends on the respective modification of EPO and/or HAS and is well known in the art (see, for example, Rose, 1994, J. Am. Chem. Soc., 116, 30, O 'Shannessay and Wichek, 1990, Analytical Biochemistry, 191, 1; Thorpe et al., 1984, Eur. J. Biochem., 140, 63; Chamov et al., 1992, J. Bioh. Chem. 267, 15916).

Regarding the methods exemplified by the present invention, Examples 2-4, especially Example 4, give a detailed description.

Step c) can be carried out in a reaction medium containing at least 10% by weight of _H2O .

In this preferred embodiment of the process according to the invention, the reaction medium contains at least 10% by weight of water, preferably at least 50%, more preferably at least 80%, for example 90% or up to 100%. According to this, the content of organic solvent can be calculated. Therefore, the reaction takes place in the aqueous phase. The preferred reaction medium is water.

An advantage of this embodiment of the process according to the invention is that toxicologically hazardous solvents do not have to be used and therefore do not have to be removed after the production process to avoid solvent contamination. Furthermore, no additional quality control is necessary for residual toxicologically hazardous solvents. Preference is given to using toxicologically non-hazardous solvents as organic solvents, such as ethanol or propylene glycol.

Another advantage of the method of the present invention is that organic solvent-induced irreversible or reversible structural changes are avoided. Therefore, polypeptides obtained according to the method of the present invention are different from polypeptides prepared in organic solvents such as DMSO.

Furthermore, it was also surprisingly found that the conjugation of HAS to drugs in aqueous solution minimizes or avoids side reactions. Thus, this embodiment of the method of the invention produces an improved product of high purity.

In the present invention, the term "hydroxyalkyl starch" refers to starch derivatives substituted with hydroxyalkyl groups. Among them, the alkyl group may be substituted. Preferred hydroxyalkyl groups contain 2-10 carbon atoms, more preferably 2-4 carbon atoms. Thus "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are preferred.

The hydroxyalkyl group of HAS contains at least one OH group.

For all embodiments of the invention, hydroxyethyl starch (HES) is most preferred.

The expression "hydroxyalkylstarch" also includes derivatives in which the alkyl groups are mono- or polysubstituted. In this regard, it is preferred that the alkyl group may be substituted by halogen, especially fluorine, or aryl as long as the HAS remains water soluble. In addition, the terminal hydroxyl group of the hydroxyalkyl group may also be esterified or etherified. In addition, the alkyl group of hydroxyalkyl starch can also be linear or branched.

Furthermore, instead of alkyl groups, linear or branched substituted or unsubstituted alkenyl groups can also be used.

In the present invention, the average molecular weight of hydroxyethyl starch may be 1-300 kDa, wherein the average molecular weight of 5-100 kDa is more preferred. The hydroxyethyl starch may further exhibit a molar substitution degree of 0.1-0.8 relative to hydroxyethyl groups, and a C2:C6 substitution ratio of 2-20.

HAS-EPO produced by the method of the invention can be purified and characterized as follows:

The separation of HAS-EPO can be carried out by known methods for purifying natural and recombinant EPO (such as size exclusion chromatography, ion exchange chromatography, RP-HPLC, hydroxyapatite chromatography, hydrophobic interaction chromatography, Example 20.8 described methods or combinations thereof).

Covalent attachment of HAS to the EPO polypeptide can be confirmed by carbohydrate composition analysis (ratio of hydroxyethylglucose to mannose present on the three N-glycosylation sites of EPO) after hydrolysis of the modified protein.

HAS modification at the N-linked oligosaccharides of EPO can be demonstrated by removing the HAS-modified N-glycans and observing the predicted shift to higher mobility using SDS-PAGE +/- Western Blotting.

The HAS modification of EPO at cysteine residues can be demonstrated based on the inability to detect the corresponding proteolytic Cys-peptide in the proteolytic fragment of the HAS modified product by RP-HPLC and MALDI/TOF-MS (Zhou et al. People, 1998, Application of capillary electrophoresis, liquid chromatogfaphy, electrospray-mass spectrometry and matrix-assisted laserdesorption/ionization-time of flight-mass spectrometry to the characterization of recombinant human erythropoietin-Electrophoresis), 55.43) Following proteolytic digestion of Cys-modified EPO, isolation of the HAS-containing fraction enabled confirmation of the corresponding peptide contained in this fraction by routine amino acid composition analysis.

All the embodiments disclosed above concerning the HAS-EPO of the present invention concerning the properties of EPO or HAS also apply to the method of the present invention for preparing HAS-EPO.

The invention also relates to a HAS-EPO obtainable by the method of the invention. Preferably, the HAS-EPO has the characteristics defined above for the HAS-EPO of the invention.

The invention also relates to the HAS-EPO according to the invention for use in a method of treatment of the human or animal body.

In addition, the present invention also relates to pharmaceutical compositions containing the HAS-EPO of the present invention. In a preferred embodiment, the pharmaceutical composition also contains at least one pharmaceutically acceptable diluent, adjuvant and/or carrier used in erythropoietin therapy.

Preferably, the pharmaceutical composition is used for treating anemia disease or hematopoietic dysfunction disease or diseases related thereto.

A "therapeutically effective amount" as used herein refers to an amount that provides a therapeutic effect for a given condition and administration regimen. Erythropoietin isoforms are preferably administered parenterally. The specific route chosen will depend on the disease being treated. Erythropoietin isoforms are preferably administered as part of a formulation containing a suitable carrier (such as human serum albumin), a suitable diluent (such as a buffer) and/or a suitable adjuvant. The required dose is an amount sufficient to increase the patient's hematocrit, and varies depending on the severity of the disease to be treated, the method of administration, and the like.

Preferably, the therapeutic object of the composition of the invention is to increase the hemoglobin value in the blood to above 6.8 mmol/l. For this purpose, the pharmaceutical composition can be administered so that the hemoglobin value increases by 0.6 mmol/l to 1.6 mmol/l weekly. If the hemoglobin value exceeds 8.7 mmol/l, preferably the treatment should be interrupted until the hemoglobin value falls below 8.1 mmol/l.

The compositions of the invention are preferably used in formulations suitable for subcutaneous or intravenous or parenteral injection. Suitable excipients and carriers for this purpose are, for example: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chlorate, polysorbate 80, HAS and water for injections. The composition may be administered three times a week, preferably twice a week, more preferably once a week, most preferably every two weeks.

Preferably, the pharmaceutical composition is administered in an amount of 0.01-10 μg/kg patient body weight, more preferably 0.1-5 μg/kg, 0.1-1 μg/kg or 0.2-0.9 μg/kg, most preferably 0.3-0.7 μg/kg, most preferably Preferably 0.4-0.6 μg/kg body weight.

Preferably, usually 10 ug-200 μg, preferably 15 μg-100 μg is administered per dose.

The present invention also relates to the application of the HAS-EPO of the present invention in the preparation of medicines for treating anemia disease or hematopoietic dysfunction disease or diseases related thereto.

According to another aspect of the present invention, the problem is solved with HAS-polypeptide conjugates (HSS-polypeptides) containing one or more hydroxyalkyl starch (HSS) molecules, wherein each HAS and polypeptide pass

a) the carbohydrate portion; or

b) Thioether coupling.

The HAS-polypeptide according to the invention has the advantage that it exhibits an increased biological stability compared to the polypeptide before conjugation. This is mainly due to the fact that HAS-polypeptides are poorly or not recognized by the clearance systems of the liver and kidneys and therefore remain in the circulation for a long time. Furthermore, since HAS is site-specifically attached, the risk of disrupting the biological activity of the polypeptide in vivo due to conjugation of HAS to the polypeptide is minimized.

The HAS-polypeptide of the present invention mainly contains two components, that is, the polypeptide and the hydroxyalkyl starch (HSS) linked thereto.

The polypeptide may be of any human or animal origin. In a preferred embodiment, the polypeptide is of human origin.

The polypeptide may be a cytokine especially erythropoietin, an antithrombin (AT) such as AT III, an interleukin especially interleukin-2, IFN-β, IFN-α, G-CSF, CSF, interleukin-6 and therapeutic Antibody.

According to a preferred embodiment, the polypeptide is antithrombin (AT), preferably ATIII (Levy JH, Weisinger A, Ziomek CA, Echelard Y, Recombinant Antithrombin: Production and Role in Cardiovascular Disorder, Seminars in Thrombosis and Hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten SM, Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Transgenically Produced Human Antithrombin: Structural and Functional Comparison to Human Plasma -Derived Antithrombin, Blood 91, 12(1998) 4661-4671; Minnema MC, Chang ACK, Jansen PM, Lubbers YTP, Pratt BM, Whittaker BG, Taylor FB, Hack CE, Friedman B, Recombinant human antithrombin III improve survival and intenuates Responses in baboonslethally challenged with Escherichia coli, Blood 95, 4 (2000) 1117-1123; Van Patten SM, Hanson EH, Bernasconi R, Zhang K, Manavalln P, Cole ES, McPherson JM, Edmunds T, Oxidation Residuith bin of Methionromine , J. Biol. Chemistry 274, 15 (1999) 10268-10276).

According to another preferred embodiment, the polypeptide is human IFN-β, in particular IFN-β1a (see Avonex®, REBIF®) and IFN-β1b (see BETASERON®).

Another preferred polypeptide is human G-CSF (granulocyte colony stimulating factor). See, e.g., Nagata et al., The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating fater, EMBO J.5:575-581, 1986; Souza et al., Recombinant human granulocyte colony-stimulating factor: effects onid uk normall and ole , Science 232 (1986) 61-65; Herman et al., Characterization, formulation, and stability of Neupogen® (Filgrastim), a recombinant human granulocyte-colony stimulating factor, see "Protein Drug Preparation, Characterization and Stability" (Formulalion, Characterization , and stability of proteindrugs), edited by Rodney Pearlman and Y.John Wang, Plenum Press, NewYork, 1996, 303-328.

With regard to erythropoietin, all embodiments disclosed above also apply here.

Preferably, the polypeptide is produced recombinantly. Production in eukaryotic or prokaryotic cells is included, preferably in mammalian, insect, yeast, bacterial cells, or any other cell type suitable for recombinant production of polypeptides. Furthermore, the polypeptides can also be expressed in transgenic animals (eg in body fluids such as milk, blood, etc.), in eggs of transgenic birds, especially poultry, preferably chickens, or in transgenic plants.

Recombinant production of polypeptides is well known in the art. It usually involves transfecting host cells with an appropriate expression vector, culturing the host cells under conditions capable of producing the polypeptide, and purifying the polypeptide from the host cells. For details, see, eg: Krystal, Pankratz, Farber, Smart, 1986, Purification of human erythropoietin tohomogeneity by a rapid five-step procedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant humanerythropoietin produced using a baculovirus vector, Blood, 74(2), 652-7; EP 640 619 B1 and EP 668 351 B1.

A polypeptide may contain one or more carbohydrate side chains attached to the polypeptide by N- and/or O-linked glycosylation, ie, the polypeptide is glycosylated. When a polypeptide is produced in eukaryotic cells, the polypeptide is usually post-translationally glycosylated. Thus, carbohydrate side chains may be attached to the polypeptide during biosynthesis in mammalian, especially human, insect or yeast cells.

HAS can be coupled directly to the polypeptide, or it can be coupled through a linker molecule. The nature of the linker molecule depends on how the HAS is linked to the polypeptide. Several adapters are commercially available (eg, from Pierce, supra). The nature of linkers and their use are described in detail below in the section on HES-polypeptide production methods.

According to a preferred embodiment of the HAS-polypeptide conjugate of the invention, the HAS is coupled to the polypeptide via a carbohydrate moiety. Preferably, the same applies when the polypeptide is an antithrombin, preferably ATIII.

In the present invention, the term "carbohydrate moiety" refers to hydroxyaldehydes or hydroxyketones, as well as their chemical modifications (see Römpp Chemielexikon, Thieme Verlag Stuttgart, Germany, 9th Edition, 9, 2281-2285, and cited here literature). Furthermore, it also refers to derivatives of naturally occurring carbohydrate moieties (eg, glucose, galactose, mannose, sialic acid, etc.). The term also includes naturally occurring carbohydrate moieties whose ring structures have been opened and chemically oxidized.

Carbohydrate moieties can be attached directly to the polypeptide backbone. Preferably, the carbohydrate moiety is part of a carbohydrate side chain. In such cases, there may also be other carbohydrate moieties between the HAS-linked carbohydrate moiety and the polypeptide backbone. More preferably, the carbohydrate moiety is the terminal moiety of a carbohydrate side chain.

In a more preferred embodiment, the HAS is coupled to the galactose residue of the carbohydrate side chain, preferably to the terminal galactose residue of the carbohydrate side chain. This galactose residue can be used for conjugation by removal of the terminal sialic acid followed by oxidation (see below).

In a more preferred embodiment, the HAS is coupled to the sialic acid residue of the carbohydrate side chain, preferably to the terminal sialic acid residue of the carbohydrate side chain.

In addition, HAS can also be coupled to peptides via thioethers. As detailed below, the S atom can be derived from any naturally or non-naturally occurring SH group attached to the polypeptide.

In a preferred embodiment, the S atom may be derived from a SH group introduced into an oxidized carbohydrate moiety of HES, preferably an oxidized carbohydrate moiety that is part of a carbohydrate side chain of a polypeptide (see below).

Preferably, the S atom in the thioether is derived from naturally occurring cysteine or from added cysteine.

In the context of the present invention, the term "added cysteine" means that the polypeptide contains a cysteine residue which is not present in the wild-type polypeptide.

In this aspect of the invention, cysteine may be an additional amino acid added at the N- or C-terminus of the polypeptide.

In addition, cysteine can also be added by substituting naturally occurring amino acids for cysteine.

The second component of the HAS-polypeptide is HAS.

In the present invention, the term "hydroxyalkyl starch" refers to starch derivatives substituted with hydroxyalkyl groups. Among them, the alkyl group may be substituted. Preferably, the hydroxyalkyl group contains 2-10 carbon atoms, more preferably 2-4 carbon atoms. Therefore, "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are preferred.

The hydroxyalkyl group of HAS contains at least one OH group.

"Hydroxyalkyl starch" also includes derivatives in which the alkyl group is mono- or polysubstituted. In this regard, it is preferred that the alkyl group is substituted by halogen, especially fluorine, or aryl as long as the HAS remains water soluble. In addition, the terminal hydroxyl group of the hydroxyalkyl group may also be esterified or etherified. In addition, the alkyl group of hydroxyalkyl starch can also be linear or branched.

In addition, linear or branched substituted or unsubstituted alkenyl groups can also be used instead of alkyl groups.

For all embodiments of the invention, hydroxyethyl starch (HES) is most preferred.

In the present invention, the average molecular weight (weight average) of hydroxyethyl starch may be 1-300 kDa, wherein the average molecular weight of 5-100 kDa is more preferred. The hydroxyethyl starch may further exhibit a molar substitution degree of 0.1-0.8 relative to the hydroxyethyl group and a C2:C6 substitution ratio of 2-20.

HAS-polypeptides may contain 1-12, preferably 1-9, 1-6 or 1-3, most preferably 1-4 HAS molecules per polypeptide molecule. The number of HAS molecules per polypeptide molecule can be determined by quantitative carbohydrate composition analysis using GC-MS after product hydrolysis and derivatization of the resulting monosaccharides (Chaplin and Kennedy (eds.), 1986, Carbohydrate Analysis: A Carbohydrate Analysis: a practical approach, Chapter 1, Monosaccharides, pp. 1-36; Chapter 2, Oligosaccharides, pp. 37-53, Chapter 3, Neutral Polysaccharides, pp. 55-96 pp.; IRL Press Practical approach series (ISBN 0-947946-44-3)).

All embodiments of the method for producing a HAS-polypeptide according to the invention disclosed below relating to the properties of the polypeptide or HAS also apply to the HAS-polypeptide according to the invention. Furthermore, all the embodiments disclosed above with respect to HAS-EPO or its preparation generally relating to peptides or HAS also apply to the HAS-polypeptides of the invention.

Hydroxyalkyl starch is an ether derivative of starch. In addition to the ether derivatives mentioned, other starch derivatives can also be used in the present invention. For example, derivatives containing esterified hydroxyl groups may be used. These derivatives may be, for example, derivatives of unsubstituted monocarboxylic or dicarboxylic acids having 2 to 12 carbon atoms, or derivatives of substituted derivatives thereof. Particularly useful are derivatives of unsubstituted monocarboxylic acids having 2 to 6 carbon atoms, especially derivatives of acetic acid. In this context, acetyl starch, butyl starch or propyl starch are preferred.

Furthermore, derivatives of unsubstituted dicarboxylic acids having 2 to 6 carbon atoms are also preferred.

For derivatives of dicarboxylic acids, it is advantageous that the second carboxyl group of the dicarboxylic acid is also esterified. In addition, monoalkyl ester derivatives of dicarboxylic acids are also suitable in the present invention.

For substituted monocarboxylic or dicarboxylic acids, the substituents may preferably be the same as those described above for substituting alkyl residues.

The technique of esterification of starch is well known in the art (see, for example, Klemm D. et al., "Comprehensive Cellulose Chemistry" (Comprehensive Cellulose Chemistry) Vol. 2, 1998, Whiley-VCH, Weinheim, New York, see in particular Chapter 4.4 , Esterification of Cellulose (ISBN3-527-29489-9).

On the other hand, the present invention relates to the method for producing hydroxyalkyl starch (HSS)-polypeptide conjugate (HSS-polypeptide), it comprises the following steps:

a) providing a polypeptide capable of reacting with the modified HAS,

b) providing a modified HAS capable of reacting with the polypeptide in step a), and

c) reacting the polypeptide of step a) with the HAS of step b), thereby producing a HAS-polypeptide comprising one or more HAS molecules, wherein each HAS passes through the polypeptide

i) the carbohydrate portion; or

ii) Thioether coupling.

The method of the present invention has the advantage of producing HAS-polypeptide conjugates exhibiting high biological activity. Furthermore, the method of the present invention also has the advantage of being able to produce effective polypeptide derivatives at low cost, since the method does not include complicated and time-consuming purification steps leading to low final yields.

Thus, in the first step of the method of the invention, a polypeptide capable of reacting with the modified HAS is provided.

The term "providing" as used in the present invention is interpreted that after each step a molecule (polypeptide in step a) and HAS in step b) can be obtained with the desired properties.

For step a), this involves purification of the polypeptide from natural sources, as well as recombinant production in host cells or organisms and, if necessary, modification of the polypeptide thus obtained.

Everything that has been said about the polypeptides as starting material according to the invention also applies to the erythropoietin as part of the HAS-polypeptide conjugates according to the invention. In this regard, the preferred embodiments disclosed above also apply to the method according to the invention.

Preferably the polypeptide is recombinantly produced. Production in eukaryotic or prokaryotic cells is included, preferably in mammalian, insect, yeast, bacterial cells or in any other cell type suitable for recombinant production of polypeptides. Furthermore, the polypeptides can also be expressed in transgenic animals (eg in body fluids such as milk, blood, etc.), in eggs of transgenic birds, especially poultry, preferably chickens, or in transgenic plants.

Recombinant production of polypeptides is well known in the art. It usually includes transfecting host cells with an appropriate expression vector, culturing host cells under conditions capable of producing polypeptides, and purifying polypeptides from host cells (Krystal, Pankratz, Farber, Smart, 1986, Purification of human erythropoietin to homogeneity by a rapidfive-step procedure, Blood, 67(1), 71-9; Quelle, Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant human erythropoietin producing using a baculovirus vector, Blood, 74(2), 652 -7; EP 640 619 B1 and EP 668 351 B1).

A polypeptide may contain one or more carbohydrate side chains attached to the polypeptide by N- and/or O-linked glycosylation, ie, the polypeptide is glycosylated. When polypeptides are produced in eukaryotic cells, the polypeptides are often post-translationally glycosylated. Thus, carbohydrate side chains may be attached to polypeptides during biosynthesis in mammalian, particularly human, insect or yeast cells, which may be cells of transgenic animals (see above).

These carbohydrate side chains can be chemically or enzymatically modified after expression in an appropriate cell, for example by removing or adding one or more carbohydrate moieties (see, e.g., Dittmar, Conradt, Hauser, Hofer, Lindenmaier, 1989, Advances in Protein Design; Bloecker, Collins, Schmidt and Schomburg, eds., GBF-Monographs, 12, 231-246, VCH Publishers, Weinheim, New York, Cambridge).

It is an object of the method of the invention to provide a HAS-polypeptide comprising one or more HAS molecules, wherein the HAS is coupled to the polypeptide via a carbohydrate moiety (i) or via a thioether (ii). Accordingly, the polypeptide provided in step a) should have the property of being capable of coupling via carbohydrate moieties and/or via thioethers. Therefore, after step a), the polypeptide may preferably contain

(1) at least one reactive group connected directly or through a linker molecule to a sulfhydryl group or a carbohydrate moiety, capable of reacting with HES or modified HES,

(2) at least one carbohydrate moiety capable of coupling to the modified HAS, and/or

(3) At least one free SH group.

For possibility (1) above, the polypeptide of step a) can preferably be obtained by coupling an appropriate linker molecule to the SH group or carbohydrate moiety of the polypeptide. 2.1 in Example 4 provides an example of such a modified polypeptide. It is important to ensure that the addition of linker molecules does not damage the polypeptide. And this is well known to those skilled in the art.

For possibility (2) above, in a preferred embodiment the modified HAS is coupled to the polypeptide via a carbohydrate moiety.

Carbohydrate moieties can be directly attached to the polypeptide backbone. Preferably, the carbohydrate moiety is part of a carbohydrate side chain. In this case, there may also be other carbohydrate moieties between the carbohydrate moiety attached to the HAS and the backbone of the polypeptide. More preferably, the carbohydrate moiety is the terminal portion of a carbohydrate side chain.

Thus, in a preferred embodiment, the modified HAS is linked (via a linker, or no linker, see below) to a carbohydrate chain which is linked to the N- and/or O-glycosylation site of the polypeptide.

However, the invention also includes polypeptides containing other carbohydrate moieties coupled to the modified HAS. Techniques for linking carbohydrate moieties to polypeptides enzymatically or by genetic engineering, and subsequent expression in appropriate cells are known in the art (Berger, Greber, Mosbach, 1986, Galactosyltransferase-dependent sialylation of complex and endo-Nacetylglucosaminidase H-treated coreN-glycans invitro, FEBS Lett., 203(1), 64-8; Dittmar, Conradt, Hauser, Hofer, Lindenmaier, 1989, Advances in Protein Design; Bloecker, Collins, Schmidt and Schomburg, eds., GBF-Monograph, 12, 231- 246, VCHP Publishers, Weinheim, New York, Cambridge).

In a preferred embodiment of the method according to the invention, the carbohydrate moiety is oxidized in order to be able to react with the modified HAS. This oxidation can be carried out chemically or enzymatically.

Methods for chemically oxidizing carbohydrate moieties of polypeptides are well known in the art and include treatment with periodate (Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).

By chemical oxidation it is in principle possible to oxidize any carbohydrate moiety, terminal or not. However, by choosing mild conditions (1 mM periodate, 0 °C, as opposed to stringent conditions: 10 mM periodate, 1 h at room temperature), it is possible to preferentially oxidize terminal carbohydrate moieties of carbohydrate side chains, such as sialic acid or galactose.

In addition, carbohydrate moieties can also be oxidized enzymatically. Enzymes for oxidizing individual carbohydrate moieties are well known in the art, eg for galactose the enzyme is galactose oxidase.

If the terminal galactose moiety is to be oxidized, if the polypeptide is produced in cells capable of linking sialic acid to carbohydrate chains, such as in mammalian cells, or in cells capable of linking sialic acid to carbohydrate chains after genetic modification, eventually one must (partially or completely) to remove terminal sialic acid. Chemical or enzymatic methods for removing sialic acid are well known in the art (Chaplin and Kennedy (Eds.), 1996, Carbohydrate Analysis: A Practical Approach, see Chapter 5, Montreuill, Glycoproteins, pp. 175-177; IRL Press Practical approach series (ISBN 0-947946-44-3)).

However, the invention also encompasses linking the carbohydrate moiety to be linked to the modified HAS to the polypeptide in step a). If it is desired to attach galactose, this can be achieved using galactosyltransferase. These methods are well known in the art (Berger, Greber, Mosbach, 1986, Galactosyltransferase-dependent sialylation of complex andendo-N-acetylglucosaminidase H-treated core N-glycans in vitro, FEBS Lett., 203(1), 64-8) .

In a most preferred embodiment, in step a), preferably after partial or complete (enzymatic and/or chemical) removal of terminal sialic acid, if necessary, by oxidizing at least one or more carbohydrate side chains of the polypeptide A terminal sugar unit (preferably galactose) is used to modify the polypeptide (see above).

Thus, it is preferred that the modified HAS is coupled to the oxidized terminal sugar unit of the carbohydrate chain, preferably to galactose.

In a further preferred embodiment (see point (3) above), the polypeptide contains at least one free SH group.

According to a preferred embodiment, the free SH group is part of a naturally occurring cysteine or an added cysteine.

Amino acid substitution methods are well known in the art (Elliott, Lorenzini, Chang, Barzilay, Delorme, 1997, Mapping of the active site of recombinant human erythropoietin, Blood, 89(2), 493-502; Boissel, Lee, Presnell, Cohen, Bunn , 1993, Erythropoietin structure-function relationships. Mutant proteins that test amodel of tertiary structure, J Biol Chem., 268(21), 15983-93)).

In the present invention, the term "added cysteine" means that the polypeptide contains a cysteine residue which is not present in the wild-type polypeptide. This can be accomplished by adding (eg, by recombinant means) a cysteine residue at the N- or C-terminus of the polypeptide, or by replacing (eg, by recombinant means) a naturally occurring amino acid with cysteine. These methods are well known to those skilled in the art (see above).

Preferably, cysteine is added by substituting a naturally occurring amino acid for a cysteine.

Preferably, the modified HAS is coupled with the added cysteine in step c).

In step b) of the method of the invention, a modified HAS capable of reacting with the polypeptide of step a) is provided.

For this purpose, HAS may preferably be modified at its reducing end. This has the advantage that the chemical reaction is easy to control and the skilled person can be sure which group of HAS is modified during the reaction. Since only one group is introduced into HAS, cross-linking of different polypeptide molecules through the multifunctional HAS molecule and other side reactions can be prevented.

Therefore, the modified HAS is able to react with the following components:

(1) at least one group that is directly or through a linker molecule connected to a sulfhydryl group or a carbohydrate moiety of a polypeptide,

(2) at least one carbohydrate moiety, preferably it is oxidized, and/or

(3) At least one free SH group.

Regarding point (1) above, the modification of HAS depends on the group attached to the polypeptide. The mechanism is well known in the art. 2.1 in Example 4 gives an example.

Regarding the above points (2) and (3), several methods of modifying HAS are known in the art. The rationale for these approaches is either to modify the reactive groups of HAS to be able to react with carbohydrate moieties or SH groups, or to couple a linker molecule to HAS containing reactive groups capable of reacting with carbohydrate moieties or SH groups. group.

For point (2), the modified HAS is capable of reacting with oxidized carbohydrate moieties, preferably with terminal sugar residues, more preferably with galactose or with terminal sialic acid.

Several methods are known to modify HAS so that it can react with oxidized preferred terminal sugar residues. As mentioned above, this modification can be introduced selectively in the reducing end region of the HES-chain. In this case, in the first step, the aldehyde group is oxidized to the lactone. These modifications include, but are not limited to, the addition of hydrazide, amino (and hydroxylamino), semicarbazide, or thiol functional groups to HAS, either directly or through a linker. These techniques are described in further detail in Examples 2-4. Furthermore, the mechanism itself is well known in the art (see, e.g. DE 196 28 705 A1; Hpoe et al., 1981, Carbohydrate Res., 91, 39; Fissekis et al., 1960, Journal of Medicinal and Pharmaceutical Chemistry, 2, 47; Frie, 1998, graduation thesis, Fachhochschule Hamburg, DE).

In the present invention, the addition of hydrazide or hydroxylamino functional groups is preferred. In this case, the selective reaction of the modified HAS with the oxidized carbohydrate moieties of the polypeptide without the lysine side chains with oxidized sugars is ensured, preferably by carrying out the reaction of step c) of the method according to the invention at pH 5.5 The residues form imines thereby causing intermolecular or intramolecular polypeptide crosslinking.

Regarding point (3), several methods are known for modifying HAS so that it can react with free SH groups. Preferably, this modification is introduced selectively in the reducing end region of the HES-chain. These methods include, but are not limited to, the addition of maleimide, disulfide, or haloacetamide functional groups to HAS. These techniques are described in further detail in Examples 2-4.

For further details on these techniques, see Chamov et al., 1992, J.Biol.Chem., 267, 15916; Thorpe et al., 1984, Eur.J.Biochem., 140, 63; Greenfield et al., 1990, Cancer Research, 50, 6600, and references cited in 1.3 of Example 2.

Table 1 lists some other possible functional groups, providing a systematic overview of possible linker molecules. Furthermore, the mechanisms themselves are well known in the art.

Several linker molecules that can be used in the present invention are known in the art or are commercially available (eg from Pierce, available from Perbio Science Deutschland GmbH, Bonn, Germany).

In step c) of the method of the present invention, the polypeptide of step a) is reacted with the HAS of step b), thereby producing a HAS-polypeptide comprising one or more HAS molecules, wherein HAS is linked to the HAS via a carbohydrate moiety or via a thioether Peptide conjugation.

In principle, the detailed method for reacting a polypeptide with a modified HAS depends on the respective modification of the polypeptide and/or HAS and is well known in the art (see, for example Rose, 1994, J. Am. Chem. Soc., 116, 30, O 'Shannessay and Wichek, 1990, Analytical Biochemistry, 191, 1; Thorpe et al., 1984, Eur. J. Bio-chem., 140, 63; Chamov et al., 1992, J. Biol. Chem. 267, 15916).

For the methods exemplified by the present invention, Examples 2-4, especially Example 4, give a detailed description.

Step c) can be carried out in a reaction medium containing at least 10% by weight of _H2O .

In this preferred embodiment of the process according to the invention, the reaction medium contains at least 10% by weight of water, preferably at least 50%, more preferably at least 80%, for example 90% or up to 100%. According to this, the content of organic solvent can be calculated. Therefore, the reaction takes place in the aqueous phase. The preferred reaction medium is water.

An advantage of this embodiment of the method according to the invention is that no toxicologically dangerous solvents have to be used and therefore no removal of these solvents after the production process is necessary to avoid solvent contamination. Furthermore, no additional quality control is necessary for residual toxicologically hazardous solvents. Preference is given to using toxicologically non-hazardous solvents as organic solvents, such as ethanol or propylene glycol.

Another advantage of the method of the present invention is that organic solvent-induced irreversible or reversible structural changes are avoided. Therefore, polypeptides obtained according to the method of the present invention are different from polypeptides prepared in organic solvents such as DMSO.

Furthermore, it was also surprisingly found that the conjugation of HAS to drugs in aqueous solution minimizes or avoids side reactions. Thus, this embodiment of the method of the invention produces an improved product of high purity.

In the present invention, the term "hydroxyalkyl starch" refers to starch derivatives substituted with hydroxyalkyl groups. Among them, the alkyl group may be substituted. Preferred hydroxyalkyl groups contain 2-10 carbon atoms, more preferably 2-4 carbon atoms. Thus "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are preferred.

The hydroxyalkyl group of HAS contains at least one OH group.

For all embodiments of the invention, hydroxyethyl starch (HES) is most preferred.

The term "hydroxyalkyl starch" also includes derivatives in which the alkyl group is mono- or polysubstituted. In this regard, it is preferred that the alkyl group may be substituted by halogen, especially fluorine, or aryl as long as the HAS remains water soluble. In addition, the terminal hydroxyl group of the hydroxyalkyl group may also be esterified or etherified. In addition, the alkyl group of hydroxyalkyl starch can also be linear or branched.

In addition, linear or branched substituted or unsubstituted alkenyl groups can also be used instead of alkyl groups.

In the present invention, the average molecular weight of hydroxyethyl starch may be 1-300 kDa, wherein the average molecular weight of 5-100 kDa is more preferred. Hydroxyethyl starch can further exhibit a molar substitution degree of 0.1-0.8 relative to hydroxyethyl groups and a C2:C6 substitution ratio of 2-20.

HAS-polypeptides produced by the methods of the invention can be purified and characterized as follows:

Isolation of HAS-polypeptides can be performed using known methods for the purification of natural and recombinant polypeptides (e.g. size exclusion chromatography, ion exchange chromatography, RP-HPLC, hydroxyapatite chromatography, hydrophobic interaction chromatography, Example 20.8 or a combination thereof).

Covalent attachment of HAS to the polypeptide can be confirmed by carbohydrate composition analysis after hydrolysis of the modified protein.

HAS modification at N-linked oligosaccharides of polypeptides can be demonstrated by removing the HAS-modified N-glycans and observing the predicted shift to higher mobility using SDS-PAGE +/- Western Blotting.

The HAS modification of a polypeptide at a cysteine residue can be confirmed based on the fact that in the proteolytic fragment of the HAS modified product, the corresponding proteolytic Cys-peptide cannot be detected by RP-HPLC and MALDI/TOF-MS (Zhou et al., 1998, Application of capillary electrophoresis, liquid chromatography, electrospray-mass spectrometry and matrix-assisted laser destruction/ionization-time of flight-mass spectrometry to the characterization of recombinant human erythropoietin-Electrophoresis), 5, 4 (13) Following proteolytic digestion of Cys-modified polypeptides, isolation of the HAS-containing fraction enables confirmation by routine amino acid composition analysis that this fraction contains the corresponding peptide.

All the embodiments disclosed above concerning the HAS-polypeptide of the invention concerning the properties of the polypeptide or HAS also apply to the method of the invention for producing the HAS-polypeptide conjugate. In addition, all the embodiments disclosed above concerning HAS-EPO or its preparation related to general peptides or HAS are also applicable to the method of the present invention for producing HAS-polypeptide conjugates.

The invention also relates to a HAS-polypeptide obtainable by the method of the invention. Preferably, the HAS-polypeptide has the characteristics described above for defining the HAS-EPO of the present invention.

According to a preferred embodiment of the present invention, the HAS used has the following general formula (I)

Wherein, R ₁ , R ₂ and R ₃ are respectively hydrogen, or straight-chain or branched-chain hydroxyalkyl groups. The term "hydroxyalkyl starch" used in the present invention is not limited to the compound (for the sake of brevity) containing the hydroxyalkyl R ₁ , R ₂ and/or R ₃ shown in the general formula (I) in the terminal carbohydrate moiety, but also refers to Compounds in which at least one hydroxyl group present anywhere in the terminal carbohydrate moiety and/or the remainder of the starch molecule HAS' is substituted by a hydroxyalkyl group R ₁ , R ₂ or R ₃ . Wherein, the alkyl group may be a linear or branched chain alkyl group, which may be suitably substituted. Preferably, the hydroxyalkyl group contains 1-10 carbon atoms, more preferably 1-6 carbon atoms, more preferably 1-4 carbon atoms, more preferably 2-4 carbon atoms. Therefore, "hydroxyalkyl starch" preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, among which hydroxyethyl starch and hydroxypropyl starch are particularly preferred, and hydroxyethyl starch is especially preferred.

HAS (preferably HES) is reactive with crosslinking compounds, the crosslinking agent is reactive with HAS (preferably HES), and is reactive with polypeptides such as those described above.

The reaction between HAS and the crosslinking compound can take place at the reducing end of HAS or at the oxidized reducing end of HAS. Therefore, HAS with the structure of general formula (I)

and/or, if the reducing end is oxidized, have the general formula (IIa)

And/or HAS of general formula (IIb) structure

Can react.

If the HAS of the general formula (I) is reacted with a crosslinking compound, the reaction preferably takes place in an aqueous medium. If HAS of the general formula (IIa) and/or (IIb) is reacted with a crosslinking compound, the reaction preferably takes place in a non-aqueous medium, for example a polar aprotic solvent or solvent mixture such as DMSO, and/or DMF.

If the HAS-polypeptide conjugate of the invention is produced by reacting a HAS derivative comprising HAS and a cross-linking compound with an oxidized carbohydrate moiety of the polypeptide, the cross-linking compound is preferably the following compound

H ₂ N-NH ₂ or or

If the HAS-polypeptide conjugate of the invention is produced by reacting a HAS derivative comprising HAS and at least one crosslinking compound with a sulfhydryl group of a polypeptide, it is preferred that the HAS is bonded at the optionally oxidized reducing end to the first crosslinking compound reaction, the crosslinking compound is preferably the following compound

H ₂ N-NH ₂ or or

And the generated HAS derivative is reacted with a second cross-linking compound capable of reacting with the HAS derivative and the sulfhydryl group of the polypeptide. For example, if the HAS derivative contains the structure -NH- as a functional group reactive with the second crosslinking compound, as detailed above, the following types of second crosslinking compounds containing functional groups F1 and F2 are especially preferred: Compound type (L) F1 F2 C Iodoalkyl N-succinimidyl ester D. bromoalkyl N-succinimidyl ester E. Maleimide N-succinimidyl ester f Pyridyldithio N-succinimidyl ester G Vinyl sulfone N-succinimidyl ester

A particularly preferred example of the first crosslinking compound is

和

and

compound

and

is particularly preferred, the following second crosslinking compound is preferred,

and

compound

is particularly preferred.

Depending on the respective reaction conditions, the solvent or solvent mixture used and/or the residue R' and/or R" of the compound R'-NH-R" reacted with HAS in aqueous medium, the hydroxyalkyl groups obtained by the above methods Starch derivatives may have the following structure (IIIa):

Accordingly, the present invention also relates to hydroxyalkyl starch derivatives having the structure of general formula (IIIa) as described above.

For example, if R' is hydrogen, the hydroxyalkyl starch derivative obtained by the above method may also have the following structure (IIIa) or (IIIb), wherein (IIIa) and (IIIb) can exist simultaneously in the reaction mixture, and has a certain equilibrium distribution:

Accordingly, the present invention also relates to hydroxyalkyl starch derivatives as described above having a structure according to the general formula (IIIb).

Furthermore, the present invention also relates to hydroxyalkyl starch derivatives present as mixtures of the structures of the above general formulas (IIIa) and (IIIb).

Depending on the reaction conditions and/or the chemical properties of the compound R'-NH-R" used for the reaction, the compound of general formula (IIIa) can contain N atoms in the flat position or the upright position, and there may also be two species with a certain equilibrium distribution. A mixture of types.

Depending on the reaction conditions and/or the chemical properties of the compound R'-NH-R" used for the reaction, the compound of general formula (IIIb) may contain a C-N double bond in E or Z conformation, or there may be two types with a certain equilibrium distribution mixture.

In some cases it may be desirable to stabilize compounds of general formula (Ilia), especially when the compounds of general formula (Ilia) are produced and used in aqueous solution. As a stabilization method, acylation of compounds of general formula (IIIa), especially when R' is hydrogen, is particularly preferred. can be generated using the general formula (IV

Any suitable reagent for the hydroxyalkyl starch derivative of a) as acylating agent.

According to a particularly preferred embodiment of the invention, the residue Ra which is part of the acylating agent is methyl. Preference is given to using carboxylic acid anhydrides, carboxylic acid halides and carboxylic acid activated esters as acylating agents.

Accordingly, the present invention also relates to hydroxyalkyl starch derivatives obtainable by the process described above, wherein the derivatives have the structure of the general formula (IVa).

The acylation is carried out at 0-30°C, preferably at 2-20°C, particularly preferably at 4-10°C.

In other cases, it may be desirable to stabilize compounds of general formula (IIIb), especially when producing and/or using compounds of general formula (IIIb) in aqueous solution. As a method of stabilization, reduction of compounds of general formula (IIIb) is particularly preferred, especially when R' is hydrogen. Any suitable reagent that yields a hydroxyalkyl starch derivative of general formula (IVb) can be used as reducing agent.

According to a particularly preferred embodiment of the invention, borohydrides, such as NaCNBH ₃ or NaBH ₄ , are used as reducing agents.

Accordingly, the present invention also relates to hydroxyalkyl starch derivatives obtainable by the process described above, wherein the derivatives have the structure of the general formula (IVb).

The reduction is carried out at 4-100°C, preferably at 10-90°C, particularly preferably at 25-80°C.

The present invention also relates to compounds (IIIa) and (IIIb), (IVa) and (IVb), (IIIa) and (VIa), (IIIa) and (IVb), (IIIb) and (IVa), (IIIb) and ( IVb), (IIIa) and (IIIb) and (IVa), (IIIa) and (IIIb) and (IVb), (IVa) and (IVb) and (IIIa), and (IVa) and (IVb) and (IIIb ), wherein (IIIa) and/or (IVa) can independently exist in a conformation in which the N atom is in a flat or upright position, and/or (IIIb) can exist in the form of a C-N double bond in the E or Z conformation.

The invention also relates to the HAS-polypeptides of the invention for use in a method of treatment of the human or animal body.

Furthermore, the present invention also relates to pharmaceutical compositions comprising the HAS-polypeptides of the present invention. In a preferred embodiment, the pharmaceutical composition also contains at least one pharmaceutically acceptable diluent, adjuvant and/or carrier used in erythropoietin therapy.

The present invention also relates to the application of the HAS-polypeptide of the present invention in the preparation of medicines for treating anemia or hematopoietic dysfunction or related diseases.

The invention is further illustrated by the following figures, tables and examples, which are in no way intended to limit the scope of the invention.

Brief description of the drawings

figure 1

Figure 1 shows the SDS-PAGE analysis of two HES-EPO conjugates

mw: molecular weight standard

Lane 1: HES-EPO generated according to Example Scheme 8: EPO coupled with hydrazide-HES 12KDL

Lane 2: HES-EPO generated according to Example Scheme 9: EPO coupled to hydroxylamine-based HES 12KDK

C: Control (unconjugated EPO); upper band represents EPO dimer

figure 2

Figure 2 demonstrates the coupling of HES to the carbohydrate moiety of the carbohydrate side chain by showing the digestion of the HAS-modified EPO form by polypeptide N-glycosidase

Lane 1: HES-EPO generated according to Example Scheme 8, N-glycosidase digestion

Lane 2: HES-EPO generated according to Example Scheme 9, N-glycosidase digestion

3 tracks: BRP EPO standard

4 lanes: BRP EPO standard, N-glycosidase digestion

mw: molecular weight standards (Bio-Rad SDS-PAGE Standards Low range, catalog number 161-0305, Bio-Rad Laboratories, Hercules, CA, USA)

image 3

Figure 3 shows the SDS-PAGE analysis of the HES-EPO conjugate produced according to Example 17.1.

Lane A: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Lane B: crude product after coupling according to Example 17.1.

Lane C: EPO starting material

Figure 4

Figure 4 shows the SDS-PAGE analysis of the HES-EPO conjugate produced according to Example 17.3.

Lane A: crude product after coupling according to Example 17.3.

Track B: EPO starting material

Lane C: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Figure 5

Figure 5 shows the SDS-PAGE analysis of the HES-EPO conjugates produced according to Examples 17.4 and 17.5.

Lane A: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Lane B: crude product after coupling according to Example 17.4.

Lane C: crude product after coupling according to Example 17.5.

Lane D: EPO starting material.

Figure 6

Figure 6 shows the SDS-PAGE analysis of the HES-EPO conjugates produced according to Examples 19.1 and 19.4.

Lane A: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Lane B: crude product after coupling according to Example 19.4.

Lane C: crude product after coupling according to Example 19.1.

Lane D: EPO starting material.

Figure 7

Figure 7 shows the SDS-PAGE analysis of the HES-EPO conjugates produced according to Examples 19.2, 19.3, 19.5 and 19.6.

Lane A: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Lane B: crude product after coupling according to Example 19.6, based on Example 13.3b).

Lane C: crude product after coupling according to Example 19.5, based on Example 13.1 b).

Lane D: Crude product after coupling according to Example 19.6, based on Example 13.3a).

Lane E: Crude product after coupling according to Example 19.5, based on Example 13.1 a).

Lane F: crude product after coupling according to Example 19.2.

Lane G: Crude product after coupling according to Example 19.3.

Track K: EPO starting material.

Figure 8

Figure 8 shows the SDS-PAGE analysis of the HES-EPO conjugates produced according to Examples 19.7, 19.8, 19.9, 19.10, 19.11 and 19.12.

Lane A: protein molecular weight standard ^Roti® -Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D);

Lane B: crude product after coupling according to Example 19.11.

Lane C: crude product after coupling according to Example 19.10.

Lane D: crude product after coupling according to Example 19.7.

Lane E: Crude product after coupling according to Example 19.8.

Lane F: Crude product after coupling according to Example 19.12.

Track G: EPO starting material.

Lane K: crude product after coupling according to Example 19.9.

Figure 9

SDS-PAGE analysis of EPO-GT-1 with mild acid treatment for 5 min = lane 2; 10 min = lane 3; 60 min = lane 4, untreated EPO = lane 1; showing migration of EPO after removal of N-glycans rate change (+N-glycosidase (PNGase)).

Figure 10

HPAEC-PAD (High Performance Anion Exchange Chromatography-Integrated Pulse Amperometric Detection) profiles of oligosaccharides isolated from untreated EPO and EPO incubated under mild acid hydrolysis conditions for 5 minutes, 10 minutes, and 60 minutes. Roman numerals I-V indicate the elution position, I = disialylated two-branched structure, II = trisialylated three-branched structure (two isomers), III = tetrasialylated four-branched structure + 2 N-acetyl Lactosamine repeat, IV = tetrasialylated four-armed structure + 1 N-acetyllactosamine repeat; V = tetrasialylated four-armed structure + no N-acetyllactosamine repeat. The elution regions of oligosaccharide structures without and with 1-4 sialic acids are indicated in brackets.

Figure 11

HPAEC-PAD of N-linked oligosaccharides after desialylation; shows the elution position of N-acetylneuraminic acid; numbers 1-9 indicate the elution positions of standard oligosaccharides; 1 = two-armed; 2 = three-armed ( 2-4 isomers), 3=tri-branched (2-6 isomers), 4=tetra-branched; 5=tri-branched+1 repeat; 6=quad-branched+1 repeat; 7=tri-branched +2 repeats; 8=four branches+2 repeats; 9=four branches+3 repeats.

Figure 12

SDS-PAGE analysis of mildly treated and untreated EPO after periodate oxidation of sialic acid residues. 1 = periodate oxidation, no acid treatment; 2 = periodate oxidation for 5 minutes, acid treatment; 3 = periodate oxidation and acid treatment for 10 minutes; 4 = periodate oxidation, no acid treatment; 5 = Non-periodate oxidized and non-acid treated BRP EPO standard.

Figure 13

HPAEC-PAD patterns of native oligosaccharides isolated from untreated EPO and from EPO incubated under mild acid hydrolysis conditions for 5 min and 10 min followed by periodate treatment. The elution regions for oligosaccharide structures without and with 1-4 sialic acids are indicated by brackets 1-5.

Figure 14

SDS-PAGE analysis of the time course of HES modification of EPO-GT-1-A: 20 μg aliquots of EPO-GT-1-A were reacted with hydroxylamine-modified HES derivative X for 30 minutes, 2, 4, 17 hours. Lane 1 = 30 minutes reaction time; Lane 2 = 2 hours reaction time; Lane 3 = 4 hours reaction time; Lane 4 = 17 hours reaction time; Lane 5 = EPO-GT-1-A without HES modification. The left panel shows that in the presence of hydroxylamine-modified HES derivatives (flow rate: 1ml.min ^-1 ) X, the mobility of EPO-GT-1-A changes as the incubation time increases: 1 lane = 30 minutes of reaction time; Lane 2 = 2 hours reaction time; Lane 3 = 4 hours reaction time; Lane 4 = 17 hours reaction time; Lane 5 = HES-modified EPO-GT-1-A. The right panel shows the analysis of the same sample after treatment with N-glycosidase.

Figure 15

SDS-PAGE analysis of the Q-Sepharose fraction of the HES-EPO conjugate. Each 1% flow-through and 1% fractions eluted at high salt concentration were concentrated with a Speed Vac concentrator and loaded onto the gel in sample buffer. EPO protein was stained with Coomassie blue. A=sample I; B=sample II; C=sample III; K=control EPO-GT-1; A1, B1, C1 and K1 denote the flow-through; A2, B2, C2 and K2 denote washing with high salt concentration detached fraction.

Figure 16a

SDS-PAGE analysis of HES-modified EPO sample A2 (see Figure 15), control EPO sample K2 and EPO-GT-1-A. To remove N-linked oligosaccharides, the EPO preparation was digested in the presence of N-glycosidase. All EPO samples showed a shift in mobility towards the low molecular weight form lacking or containing O-glycans. For HES-modified EPO sample A2, a low ratio of O-glycosylated to non-glycosylated protein bands was observed after de-N-glycosylation, and a diffuse protein band was detected around 30KDa, which may Represents the HES-modification at the sialic acid of the O-glycan residue (see asterisk-marked arrow).

Figure 16b

HES-modified EPO sample A2 (see Figure 15), control EPO sample K2 and EPO-GT-1A untreated or digested to remove N-linked oligosaccharides (see Figure 16a) in the presence of N-glycosidase after mild hydrolysis SDS-PAGE analysis. Both the high molecular weight form A2 before N-glycosidase treatment and the high molecular weight form A after treatment (see brackets with and without arrows) disappeared after acid treatment of the sample. The BRP EPO standard used for comparison was not treated with mild acid.

Figure 17

HPAEC-PAD analysis of N-linked oligosaccharide species released from HES-modified sample A, EPO-GT-1-A, and a control EPO sample (K) incubated with unmodified HES. Roman numerals I-V indicate the elution position, I = disialylated two-branched structure, II = trisialylated three-branched structure (two isomers), III = tetrasialylated four-branched structure + 2 N-acetyl Lactosamine repeat, IV = four-sialylated four-branched structure + 1 N-acetyllactosamine repeat; V = four-sialylated four-branched structure + no N-acetyllactosamine repeat; brackets indicate two, three , the elution region of tetrasialylated N-glycans, as described in Figure 10 and the legend to Figure 13 .

Figure 18

HPAEC-PAD analysis of N-linked oligosaccharide species released from HES-modified sample A, EPO-GT-1-A, and a control EPO sample (K) incubated with unmodified HES. The retention time of the standard oligosaccharide mixture is shown: Numbers 1-9 indicate the elution positions of the standard oligosaccharides: 1=two branches; 2=three branches (2-4 isomers), 3=three branches (2-6 isomer), 4=tetrabranch; 5=three-branch+1 repeat; 6=four-branch+1 repeat; 7=three-branch+2 repeat; 8=four-branch+2 repeat; 9=four-branch Branches + 3 repetitions.

Figure 19-25

Figures 19-25 represent MALDI/TOF mass spectra of enzymatically released and chemically desialylated N-glycans isolated from HES-modified EPO and control EPO preparations. The major signals ([M+Na] ⁺ ) at m/z 1809.7, 2174.8, 2539.9, 2905.0 and 3270.1 correspond to two- to four-branched complexes containing no, one or two N-acetyllactosamine repeats Type N-glycan structures with weak signals due to loss of fucose or galactose due to acid hydrolysis conditions used for desialylation of samples for MS analysis.

Figure 19

MALDI/TOF spectrum: desialylated oligosaccharides of HES-modified EPO A2.

Figure 20

MALDI/TOF spectrum: desialylated oligosaccharides of EPO GT-1-A.

Figure 21

MALDI/TOF spectrum: desialylated oligosaccharides of EPO K2.

Figure 22

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1.

Figure 23

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 by acid hydrolysis for 5 minutes.

Figure 24

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 by acid hydrolysis for 10 minutes.

Figure 25

MALDI/TOF spectrum: desialylated oligosaccharides of EPO-GT-1 by acid hydrolysis for 60 minutes.

Example

Example 1

Production of recombinant EPO

A) Production in mammalian cells

Recombinant EPO was produced in CHO cells as follows

The plasmid carrying the human EPO cDNA was cloned into a eukaryotic expression vector (pCR3, hereinafter referred to as pCREPO). Site-directed mutagenesis was performed using standard methods as described (Grabenhorst, Nimtz, Costa et al., 1998, In vivo specificity of human alpha1, 3/4-fucosyltrans ferases III-VII in the biosynthesis of Lewis(x) and sialyl Lewis(x) motifs on complex-type N-glycans-Coexpression studies from BHK-21 cells together with humanbeta-trace protein, J. Biol. Chem., 273(47), 30985-30994).

CHO cells stably expressing human EPO or its amino acid variants (e.g., Cys-29→Ser/Ala, or Cys-33→Ser/Ala, Ser-126→Ala, etc.) were generated by calcium phosphate precipitation as described (Grabenhorst et al. Human) was screened with sulfate G418. Three days after transfection, the cells were subcultured 1:5 and selected in DMEM containing 10% FBS and 1.5 g/l sulfate G418.

Using this selection procedure, typically 100-500 clones survive and are propagated for an additional 2-3 weeks in selection medium. Cell culture supernatants from confluent monolayers were then analyzed for EPO expression levels by Western blot and IEF/Western Blot.

EPO is produced from stable subclones in spinner flasks or in 21 perfusion reactors. Following published protocols, the different glycoforms of EPO containing different amounts of NeuAc (eg, 2-8, 4-10, 8-12 NeuAc residues) were separated using a combination of chromatographic methods as described below.

literature:

Grabenhorst, Conradt, 1999, The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivofunctional sublocalization and stability in the Golgi.J BiolChem., 274(51), 36107-16; Grabenhorst, Schlenke, Conz, Pohl, 1999, Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells, Glycoconj J., 16(2), 81-97; Mueller, Schlenke, Nimtz, Conradt, Hauser, 1999, Recombinant glycoprotein product quality in Ktroll-c troll-2proliferation, Biotechnology and bioengineering, 65(5), 529-536; Schlenke, Grabenhorst, Nimtz, Conradt, 1999, Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic, Cytotechnology, 30(1-3), 17-25 .

B) Production in insect cells

Recombinant human EPO was produced by the insect cell lines SF9 and SF 21 after transfection of cells with a recombinant baculovirus vector containing human EPO cDNA under the control of the polyhedrin promoter as described in the literature.

Cells grown in serum-free medium were transfected at a cell density of 2 x ¹⁰⁶ or 2 x ¹⁰⁷ cells/mL, and EPO titers in cell culture supernatants were determined daily. EPO was purified by Bluesepharose chromatography, ion exchange chromatography on Q-Sepharose and finally by RP-HPLC on C4 phase.

The purity of the product was confirmed by SDS-PAGE and N-terminal sequencing. Detailed carbohydrate structural analysis (N- and O-glycosylation) was performed following published protocols.

literature:

Grabenhorst, Hofer, Nimtz, Jager, Conradt, 1993, Biosynthesis and secretion of human in terleukin 2 glycoprotein variants from baculovirus-infecrted Sf21 cells. Charaterization of polypeptides and posttranslational modifications, Eur, 7, 9, 9 Biochem. Caslake, Burkert, Wojchowski, 1989, High-level expression and purification of a recombinant human erythropoietin producing using a baculovirus vector, Blood, 74(2), 652-7.

Example 2

Formation of reactive HES derivatives

1. SH-reactive HES

1.1 EMCH reacts with oxo-HES12KD to form SH-reactive HES 12KD B

B

Dissolve 0.144g (0.012mmol) of oxo-HES12KD (Fresenius German Patent DE 196 28 705 A1) in 0.3mL of anhydrous dimethyl sulfoxide (DMSO), and add dropwise to 34mg (0.15mmol) of EMCH (Perbio Science, Deutschland GmbH, Bonn, Germany) in a mixture in 1.5mL DMSO. After stirring at 60 °C for 19 hours, the reaction mixture was added to 16 mL of a 1:1 mixture of ethanol and acetone. The precipitate was collected by centrifugation, redissolved in 3 mL DMSO, and reprecipitated as above. SH-reactive HES 12KDB was obtained by centrifugation and vacuum drying. The coupling reaction with thiol-EPO is described in Example 3, 2.2.

Options:

All crosslinkers with hydrazide and maleimide functionalities separated by a spacer can be used in this reaction. Table 2 lists some further examples of such molecules available from Perbio Science, Deutschland GmbH, Bonn, Germany; marked with an "A". In addition, another class of crosslinkers with activated disulfide functionality instead of maleimide can also be used.

1.2 HES glycosylamine haloacetamide derivatives

a) Formation of glycosylamine ¹

1 mg of HES12KD sample was dissolved in 3 mL of saturated ammonium bicarbonate. Additional solid ammonium bicarbonate was then added to keep the solution saturated during the 120 hour incubation at 30°C. Amino-HES12KD C was desalted by direct lyophilization of the reaction mixture.

¹ Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740; Manger, Rademacher, Dwek, 1992, Biochemistry, 31, 10724-10732

b) Acylation of glycosylamine C with chloroacetic anhydride

Dissolve 1 mg of amino-HES12KD C sample in 1 mL of 1 M sodium bicarbonate and cool on ice. Solid crystals of chloroacetic anhydride (-5 mg) were added and the reaction mixture was allowed to warm to room temperature. The pH was monitored and additional base was added if the pH dropped below 7.0. After 2 hours at room temperature, a second portion of base and anhydride was added. After 6 hours, the product chloroacetamide-HES D1 (X=Cl) was desalted by mixed bed Amberlite MB-3(H)(OH) ion exchange resin.

c) Acylation of glycosylamine ² with bromoacetic anhydride

Bromoacetic anhydride was prepared as described by Thomas ³ . 1 mg amino-HES12KD C sample was dissolved in 0.1 mL dry DMF, cooled on ice, and 5 mg bromoacetic anhydride was added. The reaction mixture was allowed to warm slowly to room temperature and the solution was stirred for 3 hours. The reaction mixture was added to 1 mL of a 1:1 mixture of ethanol and acetone at -20°C. The precipitate was collected by centrifugation, redissolved in 0.1 mL DMF, and reprecipitated as above. Bromoacetamide-HESD2 (X=Br) was obtained by centrifugation and vacuum drying. The coupling reaction with thiol-EPO is described in Example 3, 1.2.

² Black, Kiss, Tull, Withers, 1993, Carbohydr. Res., 250, 195

³ Thomas, 1977, Methodes Enymol., 46, 362

d) The corresponding iodine derivatives D3 (X=I) were synthesized as described for the synthesis of D2. N-succinimidyl iodoacetate was used instead of bromoacetic anhydride and all steps were performed in the dark.

Options:

For acylation of amino groups, other activated forms of halogens, acids can be used, e.g.

--bromide or -chloride

-esters, such as N-hydroxysuccinimide esters, esters with substituted phenols (p-nitrophenol, pentafluorophenol, trichlorophenol, etc.)

Furthermore, it is also possible to use all crosslinkers which contain reactive amino groups and haloacetyl groups separated by a spacer. An example is SBAP. This molecule and some others are available from Perbio Science Deutschland GmbH, Bonn, Germany. They are marked with a "D" in Table 2. For cross-linking agents that can link amino-HES to mercapto-EPO without isolating haloacetamide-HES derivatives, see the explanation in 1.2 of Example 3.

1.3 Haloacetamide Derivatives of Amino-HES E ¹

a) Reaction of 1,4-diaminobutane with oxo-HES12KD yields amino-HES12KDE ⁴

⁴ S. Frie, Diplomarbeit, Fachhochschule Hamburg, 1998

Dissolve 1.44 g (0.12 mmol) of oxo-HES12KD in 3 mL of anhydrous dimethyl sulfoxide (DMSO), and add dropwise to 1.51 mL (15 mmol) of 1,4-diaminobutane in 15 mL of DMSO under nitrogen in the mixture. After stirring at 40° C. for 19 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated amino-HES12KDE was collected by centrifugation, redissolved in 40 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut off (cut off), Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

b) Chloroacetamide-HES12KD F1 was prepared according to the method for preparing chloroacetamide-HES12KDD1 in 1.3 above.

c) Bromoacetamide-HES12KD F2 (X=Br) was prepared according to the method for preparing bromoacetamide-HES12KD D2 in 1.3 above. The coupling reaction with thiol-EPO is described in Example 3, 1.2.

d) The corresponding iodine derivative F3 (X=I) is not isolated prior to reaction with thiol-EPO. Experiments are described in 1.1 of Example 3.

Options:

See 1.2 above.

2. CHO-reactive HES

2.1 Hydrazide-HES

a) Reaction of hydrazine with oxo-HES12KD

1.44 g (0.12 mmol) of oxo-HES12KD was dissolved in 3 mL of anhydrous dimethylsulfoxide (DMSO) and added dropwise to a mixture of 0.47 mL (15 mmol) of hydrazine in 15 mL of DMSO under nitrogen. After stirring at 40° C. for 19 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product J was collected by centrifugation, redissolved in 40 mL of water, dialyzed with 0.5% (v/v) triethylamine aqueous solution for 2 days, and dialyzed with water for 2 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany ), freeze-dried. The coupling reaction with oxidized Glyco-EPO is described in Example 4, 2.2.

b) Reaction of adipate dihydrazide with oxo-HES12KD

Dissolve 1.74 g (15 mmol) of adipate dihydrazide in 20 mL of anhydrous dimethyl sulfoxide (DMSO) at 65°C, and add the solution dissolved in 3 mL of anhydrous DMSO dropwise under nitrogen.

1.44 g (0.12 mmol) oxo-HES12KD. After stirring at 60°C for 68 hours, the reaction mixture was added to 200 mL of water. The solution containing L was dialyzed against 0.5% (v/v) triethylamine aqueous solution for 2 days and water for 2 days (SnakeSkin dialysis tubing, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized. The coupling reaction with oxidized Glyco-EPO is described in Example 4, 2.2.

Options:

In addition, derivatives in which two hydrazide groups are separated by any spacer can also be used.

3. Additional amino-HES12KD derivatives I and ^H1

Ammonolysis of D or F was performed separately as follows: 1 mg of each haloacetamide sample was dissolved in 0.1 mL of saturated ammonium carbonate. Additional solid ammonium carbonate was then added to keep the solution saturated during the 120 hour incubation at 30°C. The reaction mixture was added to 1 mL of a 1:1 mixture of ethanol and acetone at -20°C. The precipitate was collected by centrifugation, redissolved in 0.05 mL of water, and reprecipitated as above. The product amino HESH or I was obtained by centrifugation and vacuum drying. The coupling reaction with oxidized Glyco-EPO is described in Example 4, 4.1.

4. Hydroxylamine-modified HES12KD K

O-[2-(2-Aminooxy-ethoxy)-ethyl]-hydroxylamine was synthesized in two steps as described by Boturyn et al. using commercially available materials5 ^. Dissolve 1.44 g (0.12 mmol) of oxo-HES12KD in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and add dropwise to 2.04 g (15 mmol) of O-[2-(2-aminooxy-ethyl Oxy)-ethyl]-hydroxylamine in a mixture of 15 mL DMSO. After stirring at 65°C for 48 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product K was collected by centrifugation, redissolved in 40 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD molecular weight cut off, PerbioScienceDeutschland GmbH, Bonn, Germany), and lyophilized. The coupling reaction with oxidized Glyco-EPO is described in Example 4, 3.1.

Options:

Furthermore, derivatives in which two hydroxylamine groups are separated by any spacer can also be used.

⁵ Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485

5. Mercapto-HES12KD

5.1 Addition to oxo-HES12KD

1.44 g (0.12 mmol) of oxo-HES12KD was dissolved in 3 mL of anhydrous dimethylsulfoxide (DMSO) and added dropwise to a mixture of 1.16 g (15 mmol) cysteamine in 15 mL of DMSO under nitrogen. After stirring at 40° C. for 24 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product M was collected by centrifugation, redissolved in 40 mL of water, dialyzed with 0.5% (v/v) triethylamine aqueous solution for 2 days, and dialyzed with water for 2 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany ), freeze-dried. The coupling reaction with oxidized Glyco-EPO is described in Example 4, 2.1.

Options:

Derivatives in which the amino and sulfur functions are separated by any separator may also be used. Furthermore, the amino groups in the derivatives can also be replaced by hydrazine, hydrazide or hydroxylamine groups. The sulfur function can be protected eg as disulfide or trityl derivatives. But in this case, an additional deprotection step, which would release an M-like component, must be performed prior to conjugation.

5.2 Modification of Amino HES12KD E, H or I

a) Modified with SATA/SATP

1.44 g (0.12 mmol) of amino-HES12KDE E, H or I was dissolved in 3 mL of anhydrous dimethylsulfoxide (DMSO) and added dropwise to a mixture of 139 mg (0.6 mmol) of SATA in 5 mL of DMSO under nitrogen. After stirring at room temperature for 24 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product N was collected by centrifugation, redissolved in 40 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

Deprotection was performed in 50 mM sodium phosphate buffer pH 7.5 containing 25 mM EDTA and 0.5 M hydroxylamine for 2 hours at room temperature and product O was purified by dialysis against 0.1 M sodium acetate buffer pH 5.5 containing 1 mM EDTA. The coupling reaction described in 2.1 of Example 4 was carried out immediately after the deprotection reaction.

b) Modified with SPDP

1.44 g (0.12 mmol) of amino-HES12KDE E, H or I was dissolved in 3 mL of anhydrous dimethyl sulfoxide (DMSO) and added dropwise to a mixture of 187 mg (0.6 mmol) of SPDP in 5 mL of DMSO under nitrogen. After stirring at room temperature for 24 hours, the reaction mixture was added to 160 mL of a 1:1 mixture of ethanol and acetone. The precipitated product P was collected by centrifugation, redissolved in 40 mL of water, dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

Deprotection was carried out in 100 mM sodium acetate buffer (containing 100 mM sodium chloride) pH 4.5 containing 12 mg dithiothreitol (DTT) per 0.5 mL, at room temperature for 30 minutes, by using 0.1 M sodium acetate containing 1 mM EDTA Product Q was purified by dialysis against buffer pH 5.5. The coupling reaction described in 2.1 of Example 4 was carried out immediately after the deprotection reaction.

Options:

For the conversion of an amino group to a free form or a protected thiol group, several reagents can be used. After modification, the product can be isolated. Furthermore, with regard to the use of crosslinkers, they can be used directly in the coupling reaction, preferably after a purification step. Synthesis of protected forms of mercapto-HES derivatives can be used for isolation and storage of mercapto-HES derivatives. For this, all derivatives similar to SATA can be used, which contain an active ester function and a thioester function separated by any spacer. SATP (marked with "H") in Table 2 is another member of this class. Derivatives similar to SPDP may contain active ester and disulfide functions separated by any spacer. Some other members of these classes can be seen in Table 2, marked with "F". Other similar derivatives may contain an active ester function separated by any spacer and a thiol function protected as a trityl derivative.

Example 3

Coupling reaction with thiol-EPO

1. Reaction of mercapto-EPO with haloacetamide-modified SH-reactive HES

1.1 Embodiment Scheme 1

Coupling of thiol-EPO with amino-HES12KD(E, H or I) using a cross-linker containing NHS-active ester and iodoacetamide groups such as SIA ⁶

⁶ Cumber, Forrester, Foxwell, Ross, Thorpe, 1985, Methods Enrymol., 112, 207

Material

A. Borate buffer. The composition is 50 mM sodium borate, pH 8.3, 5 mM EDTA.

B. PBS, phosphate-buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.

C. Amino-HES12KD E, H or I. Prepare at 1 mg/mL in borate buffer.

D. Crosslinker stock solution: 14 mg SIA was dissolved in 1 mL DMSO.

ED-Salt ^TM dextran desalting column, 2 × 5mL column bed volume (Perbio ScienceDeutschland GmbH, Bonn, Germany)

F. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

G. Thiol EPO solution: 5 mg/mL Thiol EPO1 in borate buffer

H. Microconcentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)

method

Add 100 μL of SIA solution to 400 μL of amino HES12KDE solution, and stir at room temperature to allow it to react for 0.5 hours. Excess crosslinker was removed by centrifuging the samples at 14,000 xg for 60 minutes using a microconcentrator. After centrifugation, the sample was brought up to its initial volume with borate buffer, and this step was repeated two more times. The resulting solution was added to 1 mL of mercapto-EPO solution, and the reaction mixture was incubated at room temperature for 16 hours. At the end of the incubation, the reactivity of excess iodoacetamide was terminated by the addition of cysteine to a final concentration of 10 mM. The reaction mixture was loaded onto a desalting column equilibrated with PBS buffer, and the protein content in the fractions was monitored with Coomassie protein detection reagent. All fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophilization after overnight dialysis against water.

Options:

In this reaction, all crosslinkers containing a succinimide or sulfosuccinimide function and an iodoacetamide function separated by a spacer can be used. Some other examples can be seen in Table 2. They are marked with a "C" and are available from Perbio Science Deutschland GmbH, Bonn, Germany.

1.2 Embodiment scheme 2

Coupling of thiol EPO 1 with SH-reactive HES12KD bromoacetamide D2, F2 or iodoacetamide D37

Material

A. Phosphate buffer: composed of 100mM sodium phosphate, pH6.1, 5mM EDTA.

B. PBS, phosphate-buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.

C. SH-reactive HES12KD bromoacetamide D2. Prepared at 10 mg/mL in phosphate buffered saline.

DD-Salt ^TM dextran desalting column, 2 × 5mL column bed volume (Perbio ScienceDeutschland GmbH, Bonn, Germany)

E. Coomassie® protein assay reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

F. Thiol EPO Solution: 5 mg/mL Thiol EPO1 in Phosphate Buffered Saline

method

1 mL of SH-reactive HES12KD bromoacetamide D2 solution was mixed with 1 mL of thiol-EPO solution, and the reaction mixture was incubated at room temperature for 48 hours. At the end of the incubation, the reactivity of excess bromoacetamide was stopped by adding cysteine to a final concentration of 10 mM. The reaction mixture was loaded onto a desalting column equilibrated with PBS buffer. Fractions were monitored for protein content using Coomassie protein detection reagent, and all fractions containing protein conjugates were pooled and conjugates were obtained by lyophilization after overnight dialysis against water.

⁷ de Valasco, Merkus, Anderton, Verheul, Lizzio, Van der Zee, van Eden, Hoffmann, Verhoef, Snippe, 1995, Infect. Immun., 63, 961

Options:

Instead of the isolation of the SH-reactive HES12KD-bromoacetamide D2, the amino HES12KD (E, H, I) can be linked via a succinimide function and a bromoacetamide function with a crosslinker (see 1.1 above). SBAP is a member of this class of cross-linkers and can be seen in Table 2, marked with a "D".

2. Reaction of thiol-EPO with maleimide-modified SH-reactive HES

2.1 Embodiment Scheme 3

Use a cross-linker containing hydrazide and maleimide functional groups such as M ₂ C ₂ H to couple thiol EPO to HES12KD

Material

AM ₂ C ₂ H stock solution: 10 mg/mL M ₂ C ₂ H in DMSO, freshly prepared

B.HES12KD: 10mg/mL in 0.1M sodium acetate buffer pH5.5

C. Thiol-EPO Solution: 5 mg/mL Thiol-EPO in Phosphate/NaCl Buffer

D. Phosphate/NaCl: 0.1M Sodium Phosphate, 50mM NaCl, pH7.0

E. Micro concentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)

F. Gel filtration column: for example, Sephadex® G-200 (1.5×45cm)

G. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

H. PBS, phosphate-buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.

method

The M ₂ C ₂ H solution was added to 400 μL of the HES12KD solution to a final concentration of 1 mM, and allowed to react at room temperature for 2 hours with stirring. Excess crosslinker was removed by centrifuging the samples at 14,000 xg for 60 minutes using a microconcentrator. After centrifugation, the sample was brought up to its original volume with phosphate/NaCl buffer and this step was repeated two more times. 0.5 mL of mercapto-EPO solution was added to the M ₂ C ₂ H-modified HES12KD, and the reaction mixture was incubated at room temperature for 2 hours. At the end of the incubation, the reactivity of excess maleimide was stopped by adding cysteine to a final concentration of 10 mM. The reaction mixture was loaded onto Scphadex(R) G-200 (1.5 x 45 cm) equilibrated with PBS buffer, and fractions were collected in units of 1 mL. The protein content of the fractions was monitored with Coomassie protein detection reagent. All fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophilization after overnight dialysis against water.

Operation precautions

Hydrazone adducts are slightly unstable at extreme pH. For applications that may involve processing at low pH, we reduced hydrazones to hydrazines by treatment with 30 mM sodium cyanoborohydride in PBS buffer. For most applications, this extra step is unnecessary.

2.2 Embodiment scheme 4

Coupling of thiol EPO to maleimido-HES12KDB

Material

A. Maleimido-HES12KD B: 10mg/mL in 0.1M sodium acetate buffer pH5.5

B. Thiol-EPO Solution: 5 mg/mL Thiol-EPO in Phosphate/NaCl Buffer

C. Phosphate/NaCl: 0.1M Sodium Phosphate, 50mM NaCl, pH7.0

D. Gel filtration column: for example, Sephadex® G-200 (1.5×45cm)

E. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

F. PBS, phosphate-buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4.

method

1 mL of SH-reactive HES12KDB solution was mixed with 1 mL of thiol EPO1 solution and incubated at room temperature for 2 hours. At the end of the incubation, the reactivity of excess maleimide was terminated by adding cysteine to a final concentration of 10 mM. The reaction mixture was loaded onto Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer, and fractions were collected in units of 1 mL. The protein content of the fractions was monitored with Coomassie protein detection reagent. All fractions containing the protein conjugate were pooled and the conjugate was obtained by lyophilization after overnight dialysis against water.

2.3 Embodiment scheme 12

Coupling of thiol EPO with amino HES12KD (E, H, I) using a cross-linker containing NHS-active ester and maleimide functionality such as SMCC

Material

A. Micro concentrator: Microcon YM-10 (amicon, Milipore GmbH, Eschborn, Germany)

B.PBS, Phosphate Buffered Saline: 10mM Sodium Phosphate, 150mM NaCl, pH7.4

C. Amino HES12KD E, H or I. Prepared at 10 mg/mL in PBS buffer.

D.SMCC solution: 1mg SMCC dissolved in 50μL DMSO

ED-Salt ^TM dextran desalting column, 2 × 5mL column bed volume (Perbio ScienceDeutschland GmbH, Bonn, Germany)

F. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

G. Thiol-EPO 1 solution: 5 mg/mL Thiol-EPO in PBS buffer

method

Add 400 μl of amino HES12KDE solution to 50 μL of SMCC solution, and allow the reaction mixture to react at room temperature for 80 min and at 46 °C for 10 min. Excess crosslinker was removed by centrifuging the samples at 14000 xg for 60 minutes using a microconcentrator. Bring the volume to 450 μL with PBS buffer and repeat this step two more times. After the last centrifugation, PBS was added to the resulting solution to 450 μL, added to 1 mL of mercapto-EPO solution, and the reaction mixture was incubated at room temperature for 16 h. At the end of the incubation, the reactivity of excess maleimide was terminated by adding cysteine to a final concentration of 10 mM. The reaction mixture was loaded onto a desalting column equilibrated with PBS buffer. The protein content of the fractions was monitored with Coomassie protein detection reagent, and all fractions containing the protein conjugate were pooled, dialyzed against water overnight, and the conjugate was obtained by lyophilization.

Options:

In this reaction, it is possible to use all crosslinkers which contain a succinimide or sulfosuccinimide function and a maleimide function separated by a spacer. Some other examples of this class of molecules can be seen in Table 2, marked with "E", available from Perbio Science Deutschland GmbH, Bonn, Germany. There is another class of crosslinkers that contain activated disulfide functionality instead of maleimide functionality. These crosslinkers can also be used for coupling. However, the disulfide bond of the conjugate can be cleaved under reducing conditions. Members of this class are marked with "F" in Table 2. A third class of crosslinkers utilizes vinylsulfone functionality instead of maleimide functionality as SH-reactive groups. One member of this class is "SVSB", marked with a "G" in Table 2.

Example 4

Coupling reaction with oxidized EPO

1. Oxidation of Glyco-EPO

1.1 Oxidation of Glyco-EPO with sodium metaperiodate: Example Scheme 5

Material

A. Glyco-EPO solution: 10mg/mL Glyco-EPO in acetate buffer

B. Sodium metaperiodate solution: 10 mM or 100 mM sodium periodate in acetate buffer, freshly prepared. Store in a dark place. When using these solutions, the final concentration of sodium periodate in the oxidation mixture was 1 mM or 10 mM, respectively.

C. Acetate buffer: 0.1M sodium acetate buffer, pH5.5

D. Glycerin

E. Micro concentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)

method

All steps were performed in the dark.

Add 0.1 mL of cold sodium metaperiodate solution to 1 mL of cold Glyco-EPO solution, and carry out the oxidation reaction in the dark for 1 hour. If the Glyco-EPO to be oxidized contains sialic acid residues, the oxidation condition is 1 mM sodium periodate, 0°C. Otherwise, conditions of 10 mM sodium periodate at room temperature were used. To stop oxidation, glycerol was added to a final concentration of 15 mM and incubated at 0 °C for 5 min. Excess reagents and by-products were removed by centrifuging the product at 14,000 xg for 60 minutes using a microconcentrator. After centrifugation, the sample is brought to its original volume with the buffer used in the next modification step, such as acetate buffer. This step was repeated two more times.

1.2 Enzymatic Oxidation of Glyco-EPO: Example Scheme 6

Enzymatic oxidation of EPO has been described elsewhere (Chamow et al., 1992, J. Biol. Chem., 267, 15916-15922).

2. Coupling with hydrazine/hydrazide derivatives

2.1 Embodiment scheme 7

Oxidized Glyco-EPO was coupled with _mercapto -HES12KD M, O or Q using a cross-linker containing hydrazide and maleimide functional groups such as _M2C2H (PerbioScience, Deutschland GmbH, Bonn, Germany).

Material

AM ₂ C ₂ H stock solution: 10 mg/mL M ₂ C ₂ H in DMSO, freshly prepared

B. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL Glyco-EPO in acetate buffer

C. Thiol-HES12KD M, O or Q: 10 mg/mL in phosphate/NaCl buffer

D. Acetate buffer: 0.1M sodium acetate buffer, pH5.5

E. Phosphate/NaCl: 0.1M Sodium Phosphate, 50mM NaCl, pH7.0

F. Micro concentrator: Microcon YM-3 (amicon, Milipore GmbH, Eschborn, Germany)

G. Gel filtration column: for example, Sephadex® G-200 (1.5×45cm)

H. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

I.PBS, phosphate-buffered saline: 10mM sodium phosphate, 150mM NaCl, pH7.4

method

M ₂ C ₂ H stock solution was added to 1 mL of oxidized Glyco-EPO to a final concentration of 1 mM, and allowed to react at room temperature for 2 hours with stirring. Excess crosslinker was removed by centrifuging the samples at 14000 xg for 60 minutes using a microconcentrator. After centrifugation, the sample was brought up to its original volume with phosphate/NaCl buffer and this step was repeated two more times. 1 mL of thiol-HES12KD M, O or Q solution was added to the M ₂ C ₂ H modified Glyco-EPO, and the reaction mixture was incubated at room temperature for 16 hours. At the end of the incubation, the reactivity of excess maleimide was terminated by the addition of cysteine. The reaction mixture was loaded onto Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer, and fractions were collected in units of 1 mL. The protein content of the fractions was monitored with Coomassie protein detection reagent, and all fractions containing the protein conjugate were pooled, dialyzed against water overnight, and the conjugate was obtained by lyophilization.

Operation precautions

Hydrazone adducts are slightly unstable at extreme pH. For applications that may involve processing at low pH, we reduce the hydrazone to hydrazine by treatment with 30 mM sodium cyanoborohydride in PBS buffer. For most applications, this extra step is unnecessary.

2.2 Embodiment Scheme 8

Direct coupling of oxidized Glyco-EPO to hydrazide-HES12KD L or J

Material

A. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL Glyco-EPO in acetate buffer

B. Hydrazide-HES12KD L or J: 10mg/mL in acetate buffer

C. Acetate buffer: 0.1M sodium acetate buffer, pH5.5

D. Gel filtration column: for example, Sephadex® G-200 (1.5×45cm)

E. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

F.PBS, Phosphate Buffered Saline: 10mM Sodium Phosphate, 150mM NaCl, pH7.4

method

1 mL of hydrazide-HES12KD L or J solution was mixed with 1 mL of oxidized Glyco-EPO solution, and the reaction mixture was incubated at room temperature for 16 h with stirring. The reaction mixture was loaded onto Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer, and fractions were collected in units of 1 mL. The protein content of the fractions was monitored with Coomassie protein detection reagent, and all fractions containing the protein conjugate were pooled, dialyzed against water overnight, and the conjugate was obtained by lyophilization. The coupling results are shown in Figure 24. The observed molecular shifts demonstrate that the coupling was successful. The smear is caused by the inhomogeneity of the HES. Figure 25 demonstrates the coupling of HES to carbohydrate moieties of carbohydrate side chains.

Operation precautions

Hydrazone adducts are slightly unstable at extreme pH. For applications that may involve processing at low pH, we reduced hydrazones to hydrazines by treatment with 30 mM sodium cyanoborohydride in PBS buffer. For most applications, this extra step is unnecessary.

3. Coupling with hydroxylamine derivatives ⁸

3.1 Embodiment Scheme 9

Coupling of oxidized Glyco-EPO to hydroxylamino-HES12KD K

Material

A. Oxidized Glyco-EPO solution from 6.1.1: 5 mg/mL Glyco-EPO in acetate buffer

B. Hydroxyamino-HES12KD K: 10 mg/mL in acetate buffer

C. Acetate buffer: 0.1M sodium acetate buffer, pH5.5

D. Gel filtration column: for example, Sephadex® G-200 (1.5×45cm)

E. Coomassie® Protein Detection Reagent (Perbio Science Deutschland GmbH, Bonn, Germany)

F.PBS, Phosphate Buffered Saline: 10mM Sodium Phosphate, 150mM NaCl, pH7.4

⁸ Rose, 1994, Am. Chem. Soc., 116, 30

method

1 mL of hydroxylamino-HES12KD K solution was mixed with 1 mL of oxidized Glyco-EPO solution, and the reaction mixture was incubated at room temperature for 16 hours with stirring. The reaction mixture was loaded onto Sephadex® G-200 (1.5×45 cm) equilibrated with PBS buffer, and fractions were collected in units of 1 mL. The protein content of the fractions was monitored with Coomassie protein detection reagent, and all fractions containing the protein conjugate were pooled, dialyzed against water overnight, and the conjugate was obtained by lyophilization. The coupling results are shown in Figure 24. The molecular shift observed in lane 2 demonstrates that the coupling was successful. The smear is caused by the inhomogeneity of the HES. Figure 25 demonstrates the coupling of HES to carbohydrate moieties of carbohydrate side chains.

Example 5

Identification of galactose oxidase-treated EPO N-glycans

Recombinant EPO or partially desialylated EPO form (produced by limited weak acid hydrolysis) was incubated with galactose oxidase for 30 min at 37 °C in 0.05 M sodium phosphate buffer pH 7.0 in the presence of catalase - 4 hours. The progress of the reaction was monitored by removing a 50 μg aliquot of EPO, followed by treatment of the protein with polypeptide N-glycanase.

Before and after removal of sialic acid, the released N-linked oligosaccharides (de-N -Glycosylated peptides were monitored) for HPAEC-PAD mapping. Oxidized galactose residues in each EPO oligosaccharide were quantified based on the typical shifts observed in HPAEC-PAD, and quantification was also confirmed by MALDI/TOF MS of the oligosaccharide mixture.

Example 6

Identification of HAS-modified EPO

Separation of the HAS-modified EPO form from unreacted EPO and HAS precursor molecules is achieved by gel filtration using eg Ultrogel AcA 44/54 or similar gel filtration media. Alternatively, unreacted HAS can also be removed by immunoaffinity separation of EPO on a 4 mL column containing a monoclonal antibody conjugated to Affigel (BioRad), followed by separation of unmodified EPO by gel filtration (e.g. Medium for globulins between 20kDa-200kDa).

HAS-modified EPO was identified by SDS-PAGE analysis (using 12.5% or 10% acrylamide gels) by detection of higher molecular weights than unmodified EPO after Coomassie brilliant blue stained gels. Western Blot analysis of samples using polyclonal antibodies against recombinant human EPO can also identify the higher molecular weight of HAS-modified EPO polypeptides.

EPO-type N-glycan modification was demonstrated by successful removal from EPO protein with polypeptide N-glycanase (recombinant N-glycosidase, from Roche, Germany, using 25 units/mg EPO protein, 37°C for 16 hours); SDS -PAGE analysis showed that the migration position of EPO protein to N-glycosidase-treated unmodified EPO typically shifted by about 20KDa.

The migration position of de-N-glycosylated products compared with unreacted de-N-glycosylated EPO was detected by SDS-PAGE, and it was confirmed that the mono-desialylated and galactose oxidase-treated EPOO-glycans on Ser Modifications at 126. When necessary, modified EPO was fractionated by RP-HPLC on C8-phase before SDS-PAGE analysis. HAS O-glycan modification of EPO was analyzed by β-removal of O-glycans and detection of de-O-glycosylated forms of EPO by Western blot using anti-recombinant human EPO polyclonal antibody.

Example 7

Quantification of EPO and modified EPO forms

EPO was quantified using UV measurements as described in the European Pharmacopoeia (2000, Erythropoietini solutio concentrata, 1316, 780-785) and compared to the international BRP reference EPO standard. In addition, EPO concentration can also be determined based on RP-HPLC analysis using RP-C4-column and absorbance at 254 nm (calibrated with 20, 40, 80, 120 μg BRP standard EPO reference preparation).

Example 8

In Vitro Biological Activity of HES Modified Recombinant Human EPO

The activity of purified HES-modified EPO was tested using the erythropoietin bioactivity assay as described by Krystal [Krystal, 1984, Exp. Heamatol., 11, 649-660].

Anemia was induced in NMRI mice by phenylhydrazine hydrochloride treatment and splenocytes were collected and used as described [Fibi et al., 1991, Blood, 77, 1203ff.]. Dilutions of EPO were incubated with 3 x ¹⁰⁵ cells/well in a 96-well microtiter plate. After incubation for 24 hours at 37°C in a humidified atmosphere (5% CO ₂ ), cells were labeled with 1 μCi ³ H-thymidine per well for 4 hours. Incorporated radioactivity was measured by liquid scintillation counting. The international reference EPO standard (BRP-standard) was used for comparison.

In addition, the biological activity of EPO can also be measured by an in vitro assay using the EPO-sensitive cell line TF-1 (Kitamura et al., [J. cellPhys., 140. 323-334]). Growth factors were washed out of exponentially growing cells and incubated for an additional 48 hours in the presence of serially diluted EPO. Cell proliferation was estimated using the MTT reduction assay described by Mosmann [Mosman, 1983, J. Immunol. Methods, 65, 55-63].

Example 9

In vivo activity assay of EPO and HAS-modified EPO forms

In vivo activity assays were performed in normocytic mice by measuring the increase in reticulocytes 4 days after the animals received predetermined doses of EPO or modified EPO forms. Assays were performed using the BRPEPO standard calibrated with the WHO EPO standard in the polycythemia mouse assay. EPO samples were diluted with phosphate buffered saline containing 1 mg/ml bovine serum albumin (Sigma).

Each animal was injected subcutaneously with 0.5 ml of EPO assay solution in Dulbecco's buffered saline (equivalent to 100, 80, 40 or 20 IU/ml BRP standard EPO protein amount). A blood sample was collected 4 days after the injection, and the reticulocytes were stained with acridine orange; the reticulocytes were quantified by counting a total of 30,000 blood cells by flow cytometry within 5 hours after blood sample collection (see European Pharmacopoeia, 2000, Erythropoietini solutio concentrata, 1316 , 780-785 and European Pharmacopoeia (1996/2000, Annex 2002)).

Example 10

In vivo half-life determination

Rabbits were injected intravenously with the indicated amounts of unmodified or HAS-modified EPO forms. Blood samples were collected at designated times and serum was prepared. Serum erythropoietin levels were determined by in vitro bioassay or by EPO-specific commercial ELISA.

Example 11

In vivo pharmacokinetics

Mice: Each animal received 300IU EPO/kg subcutaneously. The hematocrit of each animal was measured 7 days after treatment. A significant increase in hematocrit was observed in 9 of all animals treated with modified EPO, an expected result due to the relatively short half-life of untreated EPO. The mean change in hematocrit of the modified EPO-treated group was significantly different from that of the untreated EPO group and the control group.

Rabbits: Rabbits were treated with a single dose of unmodified or HAS-modified EPO equivalent to 200 or up to 800 ng/kg body weight. Blood samples were analyzed after 2, 6, 16, 24, 48 hours using a commercial EPO-specific ELISA for determination of plasma concentrations. The mean plasma EPO concentration was determined as described (Zettlmissl et al., 1989, J. Biol. Chem., 264, 21153-21159), and the mean initial half-life (α-phase) and terminal half-life (β-phase) were calculated from ELISA values. ).

literature:

Sytkowski, Lunn, Risinger and Davis, 1999, An Erythropoietin Fusion Protein Composed of Identical Repeating Domains Exhititis Enhanced Biological Properites, J. Biol. Chem., 274, 24773-24778.

Example 12

Evaluation of in vitro biological activity of HES-modified recombinant human IL-2

Modified IL2 was recovered by gel filtration on Ultrogel AcA 54. Aliquots of the corresponding fractions were sterile filtered and the bioactivity of IL2 was assayed using the IL2-dependent murine CTLL-2 cell line [Gillis, Ferm, On and Smith, 1978, J. Immunol., 120, 2027-2032]. Activity correlates to the international reference IL2 standard preparation.

Example 13

Formation of hydroxyethyl starch derivatives by reductive amination of unoxidized reducing ends

Example 13.1 Reaction of hydroxyethyl starch with 1,3-diamino-2-hydroxypropane

a) To a solution of 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) in 5 ml of water was added 0.83 mmol of 1,3-diamino-2-hydroxypropane and 50 mg of sodium cyanoborohydride NaCNBH ₃ . The mixture obtained was incubated at 80°C for 17 hours. The reaction mixture was added to 160 mL of a cold 1:1 mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD molecular weight cut off, Perbio Science Deutschland GmbH, Bonn, D), and freeze-dried.

b) Add 0.83 mmol 1,3-diamino-2-hydroxypropane and 50 mg sodium cyanoborohydride NaCNBH ₃ to a solution of 200 mg hydroxyethyl starch, and incubate at 25° C. for 3 days.

Example 13.2 Reaction of hydroxyethyl starch with 1,2-dihydroxy-3-aminopropane

a) To a solution of 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) in 5 ml of water was added 0.83 mmol of 1,2-dihydroxy-3-aminopropane and 50 mg of sodium cyanoborohydride NaCNBH ₃ . The mixture obtained was incubated at 80°C for 17 hours. The reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD molecular weight cut-off, PerbioScience Deutschland GmbH, Bonn, D), and lyophilized.

As described by G.Avigad, Anal.Biochem.134 (1983) 449-504, the 1,2-diol in the reaction product was oxidatively cleaved with periodate to produce aldehyde, and the 1,2-diol was indirectly confirmed by quantification of aldehyde. Reaction of hydroxy-3-aminopropane with HES.

b) Add 0.83 mmol 1,2-dihydroxy-3-aminopropane and 50 mg sodium cyanoborohydride NaCNBH ₃ to a solution of 200 mg hydroxyethyl starch, and incubate at 25° C. for 3 days.

Example 13.3 Reaction of hydroxyethyl starch and 1,4-diaminobutane

a) To a solution of 200mg hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) in 5ml water was added 0.83mmol 1,4-diaminobutane and 50mg sodium cyanoborohydride _NaCNBH3 . The mixture obtained was incubated at 80°C for 17 hours. The reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD molecular weight cut off, Perbio Science Deutschland GmbH, Bonn, D), and freeze-dried.

b) Add 0.83 mmol 1,4-diaminobutane and 50 mg sodium cyanoborohydride NaCNBH ₃ to a solution of 200 mg hydroxyethyl starch, and incubate at 25° C. for 3 days.

Example 13.4 Reaction of hydroxyethyl starch with 1-mercapto-2-aminoethane

a) To a solution of 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) in 5 ml of water was added 0.83 mmol of 1-mercapto-2-aminoethane and 50 mg of sodium cyanoborohydride NaCNBH ₃ . The mixture obtained was incubated at 80°C for 17 hours. The reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed with water for 4 days (snakeSkin dialysis tube, 3.5 KD molecular weight cut-off, PerbioScience Deutschland GmbH, Bonn, D), and freeze-dried.

b) Add 0.83 mmol 1-mercapto-2-aminoethane and 50 mg sodium cyanoborohydride NaCNBH ₃ to a solution of 200 mg hydroxyethyl starch, and incubate at 25° C. for 3 days.

Example 14:

Formation of hydroxyethyl starch derivatives by coupling with non-oxidized reducing ends

Example 14.1: Reaction of hydroxyethyl starch with carbohydrazide

0.96g HES18/0.4 (MW=18,000D, DS=0.4) was dissolved in 8ml 0.1M sodium acetate aqueous buffer pH5.2, and 8mmol carbohydrazide (Sigma Aldrich, Taufkirchen, D) was added. After stirring at 25°C for 18 hours, the reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation, redissolved in 40 ml of water, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD molecular weight cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.

Example 14.2: Reaction of hydroxyethyl starch with adipate dihydrazide

0.96g HES18/0.4 (MW=18,000D, DS=0.4) was dissolved in 8ml 0.1M sodium acetate aqueous buffer pH5.2, and 8mmol adipic dihydrazide (Lancaster Synthesis, Frankfurt/Main, D) was added. After stirring at 25°C for 18 hours, the reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation, redissolved in 40 ml of water, dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.

Example 14.3: Reaction of hydroxyethyl starch with 1,4-phenylene-di-3-thiosemicarbazide

Dissolve 0.96g HES18/0.4 (MW=18,000D, DS=0.4) in 8ml 0.1M sodium acetate aqueous buffer pH5.2, add 8mmol 1,4-phenylene-di-3-thiosemicarbazide (Lancaster Synthesis, Frankfurt/Main, D). After stirring at 25°C for 18 hours, 8 ml of water was added to the reaction mixture, and the suspension was centrifuged at 4,500 rpm for 15 minutes. The clear supernatant was decanted and then added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation, redissolved in 40 ml of water, and centrifuged at 4,500 rpm for 15 minutes. The clarified supernatant was dialyzed against water for 3 days (SnakeSkin dialysis tubing, 3.5 KD molecular weight cut off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.

Example 14.4: Reaction of hydroxyethyl starch with O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine

O-[2-(2-Aminooxy-ethoxy)-ethyl]-hydroxylamine was synthesized in two steps from commercially available materials as described by Boturyn et al. Tetrahedron 53 (1997) 5485-5492.

Dissolve 0.96g HES18/0.4 (MW=18,000D, DS=0.4) in 8ml 0.1M sodium acetate aqueous buffer pH5.2, add 8mmol O-[2-(2-aminooxy-ethoxy)- Ethyl]-hydroxylamine. After stirring at 25°C for 18 hours, the reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitated product was collected by centrifugation, redissolved in 40 ml of water, dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, D), and lyophilized.

Example 15

Formation of hydroxyethyl starch derivatives by reaction with oxidized reducing ends

Example 15.1 Reaction of hydroxyethyl starch and carbohydrazide

Dissolve 0.12 mmol of oxo-HES 10/0.4 (MW = 10,000 D, DS = 0.4, prepared according to DE196 28 705 A1) in 3 ml of anhydrous dimethyl sulfoxide (DMSO) and add dropwise to 15 mmol of carbon Hydrazide (Sigma Aldrich, Taufkirchen, D) in a mixture of 15 ml DMSO. After stirring at 65°C for 88 hours, the reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed with water for 4 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbioscience Deutschland GmbH, Bonn, D), and freeze-dried.

Example 15.2 Reaction of hydroxyethyl starch with 1,4-phenylene-di-3-thiosemicarbazide

Dissolve 0.12 mmol of oxo-HES 10/0.4 (MW = 10,000 D, DS = 0.4, prepared according to DE196 28 705 A1) in 3 ml of anhydrous dimethylsulfoxide (DMSO) and add dropwise to 15 mmol under nitrogen , 4-phenylene-di-3-thiosemicarbazide (Lancaster Synthesis, Frankfurt/Main, D) in a mixture of 15 ml DMSO. After stirring at 65°C for 88 hours, the reaction mixture was added to 160 mL of a cold 1:1 (v/v) mixture of acetone and ethanol. The precipitate was collected by centrifugation, dialyzed with water for 4 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, D), and freeze-dried.

Example 15.3 Reaction of hydroxyethyl starch and hydrazine

H ₂ N-NH ₂

Dissolve 1.44 g (0.12 mmol) of oxo-HES 10/0.4 (MW = 10,000 D, DS = 0.4, prepared according to DE 196 287 05 A1) in 3 ml of anhydrous dimethyl sulfoxide (DMSO), gradually under nitrogen Add dropwise to a mixture of 0.47ml (15mmol) hydrazine in 15ml DMSO. After stirring at 40° C. for 19 hours, the reaction mixture was added to 160 mL of a 1:1 (v/v) mixture of ethanol and acetone. The precipitated product was collected by centrifugation, redissolved in 40 mL of water, dialyzed with 0.5% (v/v) triethylamine aqueous solution for 2 days, and dialyzed with water for 2 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany) , freeze-dried.

The reaction of embodiment 15.4 hydroxyethyl starch and hydroxylamine

O-[2-(2-Aminoxy-ethoxy)-ethyl]-hydroxylamine was synthesized in two steps from commercially available materials as described by Boturyn et al. (Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997 , Tetrahedron, 53, 5485).

Dissolve 1.44 g (0.12 mmol) of oxo-HES 10/0.4 (MW = 10,000 D, DS = 0.4, prepared according to DE 196 287 05 A1) in 3 ml of anhydrous dimethyl sulfoxide (DMSO), gradually under nitrogen Add dropwise to a mixture of 2.04 g (15 mmol) O-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine in 15 ml DMSO. After stirring at 65°C for 48 hours, the reaction mixture was added to 160 mL of a 1:1 (v/v) mixture of ethanol and acetone. The precipitated product was collected by centrifugation, redissolved in 40 mL of water, dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

Example 15.5 Reaction of hydroxyethyl starch and adipate dihydrazide

1.74g (15mmol) of adipic acid dihydrazide was dissolved in 20ml of anhydrous dimethylsulfoxide (DMSO) at 65°C, and 1.44g ( 0.12 mmol) Oxo-HES 10/0.4 (MW=10,000D, DS=0.4, prepared according to DE 196 28 705 A1). After stirring at 60°C for 68 hours, the reaction mixture was added to 200 mL of water. The solution containing the reaction product was dialyzed against 0.5% (v/v) triethylamine aqueous solution for 2 days and water for 2 days (SnakeSkin dialysis tubing, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

Example 15.6 Reaction of hydroxyethyl starch and 1,4-diaminobutane

Dissolve 1.44 g (0.12 mmol) of oxo-HES 10/0.4 (MW = 10,000 D, DS = 0.4, prepared according to DE 196 287 05 A1) in 3 ml of anhydrous dimethyl sulfoxide (DMSO), gradually under nitrogen Add dropwise to a mixture of 1.51 ml (15 mmol) 1,4-diaminobutane in 15 ml DMSO. After stirring at 40° C. for 19 hours, the reaction mixture was added to 160 mL of a 1:1 (v/v) mixture of ethanol and acetone. The precipitated amino-HES10KD/0.4 was collected by centrifugation, redissolved in 40ml of water, dialyzed with water for 4 days (SnakeSkin dialysis tube, 3.5KD molecular weight cut-off, Perbio Science Deutschland GmbH, Bonn, Germany), and lyophilized.

Example 16 Oxidation of Erythropoietin

Oxidized erythropoietin was produced as described in Example 20. EPO-GT-1-A described in Example 20.11(c) was used as oxidized erythropoietin (EPO-GT-1 treated with mild periodate oxidation without acid hydrolysis).

Example 17:

Coupling of Hetastarch Derivatives and Erythropoietin Oxidized in Example 4

Example 17.1 Reaction of Oxidized Erythropoietin with the Reaction Product of Example 14.1

20 mMPBS buffer containing oxidized EPO (1.055 μg/μl) was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. To 19 µl of the EPO solution was added 18 µl of the HES derivative solution (MW 18kD; 18.7 µg/µl in 0.1 M sodium acetate buffer pH 5.2) produced according to Example 14.1, and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 3. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 17.2 Reaction of oxidized erythropoietin with the reaction product of Example 14.3

20 mMPBS buffer containing oxidized EPO (1.055 μg/μl) was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. To 19 μl of the EPO solution was added 18 μl of the HES derivative solution (MW 18 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer pH 5.2) produced according to Example 14.3, and the mixture was incubated at 25° C. for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions.

Example 17.3 Reaction of Oxidized Erythropoietin with the Reaction Product of Example 14.4

20 mMPBS buffer containing oxidized EPO (1.055 μg/μl) was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. To 19 μl of the EPO solution was added 18 μl of the HES derivative solution (MW 18kD; 18.7 μg/μl in 0.1M sodium acetate buffer pH 5.2) produced according to Example 14.4, and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 4. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 17.4 Reaction of Oxidized Erythropoietin with the Reaction Product of Example 15.1

20 mMPBS buffer containing oxidized EPO (1.055 μg/μl) was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. To 19 μl of the EPO solution was added 18 μl of the HES derivative solution (MW 10 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer pH 5.2) produced according to Example 15.1, and the mixture was incubated at 25° C. for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 5. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Reaction of the oxidized erythropoietin of Example 17.5 with the reaction product of Example 15.2

20 mMPBS buffer containing oxidized EPO (1.055 μg/μl) was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. To 19 μl of the EPO solution was added 18 μl of the HES derivative solution (MW 10 kD; 18.7 μg/μl in 0.1 M sodium acetate buffer pH 5.2) produced according to Example 15.1, and the mixture was incubated at 25° C. for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 5. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 18 Formation of thiol-EPO by reduction of erythropoietin

241.5 μg of erythropoietin (EPO-GT-1, see Example 20) was incubated in 500 μl of 0.1 M sodium borate buffer, 5 mM EDTA, 10 mM DTT (Lancaster, Morcambe, UK), pH 8.3 at 37° C. for 1 hour . Use VIVASPIN 0.5ml concentrator, 10KD MWCO (VIVASCIENCE, Hannover, Germany), remove DTT by centrifugation at 13,000rpm, then wash with borate buffer 3 times, wash with phosphate buffer (0.1M, 9.15M NaCl, 50mM EDTA, pH7.2) and washed twice.

Example 19:

Coupling of hydroxyethyl starch derivatives and mercapto-erythropoietin with cross-linking compounds

In each of the following examples, N-([alpha]-maleimidoacetoxy)succinimide ester (AMAS) was used as the crosslinking compound.

Example 19.1 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 14.1

To 50 nmol of the HES derivative produced according to Example 14.1 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) Solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 6. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.2 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 14.2

To 50 nmol of the HES derivative produced according to Example 14.2 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) Solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 7. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.3 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 14.3

To 50 nmol of the HES derivative produced according to Example 14.3 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) Solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 7. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.4 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 14.4

To 50 nmol of the HES derivative produced according to Example 14.4 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) Solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 6. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.5 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 13.1

50 nmol HES derived from Example 13.1 produced according to Example 13.1 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) incubated at 80° C. for 17 hours and 25° C. for 3 days 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO was added to the mixture. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 7. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.6 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 13.3

To 50 nmol HES produced according to Example 13.3 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) incubated at 80° C. for 17 hours and 25° C. for 3 days To the derivatives was added 10 μl of a solution of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), centrifugal filter with 13,000rpm, and wash with phosphate buffer saline 4 times each 30 minutes, remove remaining AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 7. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.7 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.1

To 50 nmol of the HES derivative produced according to Example 15.1 and dissolved in 200 μl of sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO solution in. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.8 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.2

To 50 nmol of the HES derivative produced according to Example 15.2 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in DMSO solution in. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.9 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.3

To 50 nmol of the HES derivative produced according to Example 15.3 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufklrchen, D) in DMSO solution in. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.10 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.4

To 50 nmol of the HES derivative produced according to Example 15.4 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.11 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.5

To 50 nmol of the HES derivative produced according to Example 15.5 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

Example 19.12 Reaction of mercapto erythropoietin with the reaction product and cross-linking compound of Example 15.6

To 50 nmol of the HES derivative produced according to Example 15.6 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) was added 10 μl of 2.5 μmol AMAS (Sigma Aldrich, Taufkirchen, D) in solution in DMSO. The clear solution was incubated at 25°C for 80 minutes and at 40°C for 20 minutes. Use VIVASPIN 0.5ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany), with 13,000rpm centrifugal filtration, and wash with phosphate buffer saline 4 times each 30 minutes, remove AMAS.

To the remaining solution was added 15 µg of thiol EPO produced according to Example 18 (1 µg/µl in phosphate buffer), and the mixture was incubated at 25°C for 16 hours. After lyophilization, the crude product was analyzed by SDS-PAGE using NuPAGE 10% Bis-Tris Gels/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the Invitrogen instructions. Gels were stained overnight with Roti-Coomassie blue staining reagent (Roth, Karlsruhe, D).

The experimental results are shown in Figure 8. Migration of protein bands to higher molecular weights indicates successful coupling. The increase in bandwidth is due to the molecular weight distribution of the HES derivatives used and the number of HES derivatives attached to the protein.

The preparative production of embodiment 20HES-EPO conjugate

overview

HES-EPO conjugates were synthesized as follows: HES derivatives (average molecular weight 18,000 Daltons; degree of hydroxyethyl substitution 0.4) were oxidized with the oligosaccharide chain part of recombinant human EPO (mild periodate) Sialic acid residue coupling. According to carbohydrate structure analysis, the introduced modifications did not affect the structural integrity of the core oligosaccharide chains, as MALDI/TOF-MS of the weakly acid-treated HES-modified glycans showed intact neutral N-acetyllactosamine-type chains, It was indistinguishable from the chain seen in the unmodified EPO product. The results obtained show that, for EPO preparations modified without prior partial removal of sialic acid, at least 3 modified HES residues are linked per EPO molecule. EPO variants lacking approximately 50% of the sialic acid residues in the aforementioned proteins showed similarly high apparent molecular weight mobility in SDS-PAGE (60-110 KDa versus 40 KDa for the BRPEPO standard). HES-modified EPO is stable under standard ion-exchange chromatography conditions at room temperature and pH 3-10.

EPO bioassay was carried out in a normal erythrocyte line of mice, according to the European Pharmacopoeia, the protein was determined by ultraviolet absorption value, and the EPO protein was determined by RP-HPLC and calibrated by the BRP EPO standard product. The specific activity (IU/mg) of EPO is 2.5-3.0 times higher than the reference standard.

Example 20.1 Materials and methods

(a) Release of N-linked oligosaccharides by N-glycosidase digestion

Samples were incubated overnight at 37°C with 25 units of recombinant PNGase F (according to the manufacturer's instructions from Roche Diagnostics, Germany). Complete digestion was monitored based on the specific mobility shift of the protein in SDS-PAGE. Released N-glycans were isolated from the polypeptides by adding 3 volumes of cold 100% ethanol and placing at -20°C for at least 2 hours (Schroeter S et al., 1999). Precipitated protein was removed by centrifugation at 13000 rpm for 10 min at 4°C. The pellet was then washed two more times with 500 μl of ice-cold 75% ethanol. Oligosaccharides in the pooled supernatants were dried in a vacuum centrifuge (Speed Vac concentrator, Savant Instruments Inc., USA). Glycan samples were desalted prior to use using Hypercarb columns (25 mg or 100 mg HyperCarb) as follows: the column was washed 3 times with 500 μl of 80% acetonitrile (v/v) in 0.1% TFA, followed by 3 washes with 500 μl of water. The sample was diluted with water to a final volume of 300μl-600μl, then applied to the column, and then the column was fully washed with water. Oligosaccharides were eluted with 1.2 ml (25 mg column; 1.8 ml for 100 mg column) of 25% acetonitrile in water containing 0.1% trifluoroacetic acid (v/v). Eluted oligosaccharides were neutralized with 2M _NH4OH and dried in a Speed Vac concentrator. In some cases, N-glycosidase-released oligosaccharides were desalted by absorbing <100 μg of the total (glyco)protein sample from the digestion mixture onto a 100 mg Hypercarb column.

(b) Analysis of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/TOF-MS)

Analysis of native desialylation using Bruker ULTRAFLEX time-of-flight (TOF/TOF) instrument: 2,5-dihydroxybenzoic acid as UV-absorbing material in positive and negative ion modes and reflectron in both modes of oligosaccharides. For MS-MS analysis, precursor ions are selected for laser-induced dissociation (LID), and the fragment ions obtained are separated using the instrument's second TOF stage (LIFT). 1 µl of the sample solution at a concentration of approximately 1-10 pmol·µl ^-1 was mixed with an equal amount of the respective matrix. The mixture was spotted onto a stainless steel target and dried at room temperature before analysis.

Example 20.2 Preparation and Identification of Recombinant Human EPO (EPO-GT-1)

EPO was expressed by recombinant CHO cells as described (Mueller PP et al., 1999, Dorner AJ et al., 1984) and these preparations were identified according to the method described in the European Pharmacopoeia (Ph. Eur. 4, Monography 01/2002: 1316: Erythropoietin concentrated solution). The sialic acid content of the final product was 12 nMol per nMol protein (+/- 1.5 nMol). The structures of N-linked oligosaccharides were determined by HPAEC-PAD and MALDI/TOF-MS as described (Nimtz et al., 1999, Grabenhorst, 1999). The EPO preparations obtained contained di-, tri- and tetra-sialylated oligosaccharides (2-12%, 15-28% and 60-80%, respectively, with sulfated and pentasialylated chains present in small amounts). The total glycosylation profile of the EPO preparation was similar to the international BRPEPO standard preparation.

The isoelectric focusing model of recombinant EPO was comparable to the international BRP reference EPO standard preparation, indicating the corresponding isoform. 25% of EPO proteins lack O-glycosylation at Ser126 of the polypeptide chain.

Example 20.3 Preparation of Partially Desialylated EPO Form

EPO GT-1 protein (2.84mg/ml) was heated to 80°C in 20mM sodium phosphate buffer pH 7.0, and then 100μl 1N H ₂ SO ₄ was added per 1ml of EPO solution; the incubation was continued for 5 minutes, 10 minutes, 60 minutes, respectively. minutes, produce EPO preparations with different degrees of sialylation. Oligosaccharides containing 0-4 sialic acids were quantified after release of oligosaccharides with polypeptide N-glycosidase, and separation of N-linked chains was performed by desalting with a Hypercarb column (25 mg HyperSep Hypercarb; Thermo-Hypersil-Keystone, UK). The EPO preparation was neutralized by adding 1N NaOH, frozen in liquid nitrogen, and stored at -20°C until used in the next step.

Example 20.4 Periodate Oxidation of the Sialylated EPO Form

Add 1.5ml 0.1M sodium acetate buffer pH5.5 to 10mg untreated or weakly acid-treated EPO dissolved in 3.5ml 20mM sodium phosphate buffer pH7.0, cool the mixture to 0°C in an ice bath; add 500μl 10mM sodium periodate, and the reaction mixture was kept at 0°C in the dark for 60 minutes. Add 10 μl of glycerol and continue to incubate in the dark for 10 minutes. Partially oxidized EPO forms were separated from the reagents using a VIVASPIN concentrator (10,000 MWCO, PES Vivasciernce AG, Hannover, Germany) desalted at 3000 rpm in a laboratory centrifuge equipped with a fixed-angle rotor according to the manufacturer's recommendations. After freezing in liquid nitrogen, EPO preparations were stored at -20°C in a final volume of 4 ml.

A 100 [mu]g aliquot of the partially oxidized EPO preparation was N-glycosidase treated and oligosaccharides were separated using a Hypercarb column as described above. Oligosaccharides were desialylated by mild acid treatment and analyzed by HPAEC-PAD with retention times comparable to authentic standard oligosaccharides as described (Nimtz et al., 1990 and 1993).

Example 20.5 Reduction of EPO disulfide with dithioerythritol

Incubate 5 mg of EPO-GT-1 in 5 ml of 0.1 M Tris/HCl buffer pH 8.1 in the presence of 30 mM dithioerythritol (DTT) for 60 min at 37 °C; remove at 4 °C using a Vivaspin concentrator DTT, 4 cycles of buffer exchange. The final reduced EPO preparation was frozen in liquid nitrogen and stored at -20°C in 50 mM sodium acetate buffer pH 5.5.

Example 20.6 EPO protein assay

Quantitative determination of EPO protein was carried out as follows: UV absorbance at 280 nm was measured in cuvettes with a diameter of 1 cm according to the European Pharmacopoeia (European Pharmacopoeia 4, Monograph 01/2002: 1316: Concentrated Erythropoietin Solutions). In addition, EPO can also be quantified by RP-HPLC method using RP-C4 column (Vydac Protein C4, catalog number 214TP5410, Grace Vydac, Ca, US); HPLC method is calibrated with erythropoietin BRP1 reference standard (European Pharmacopoeia, Conseil de l'Europe B.P. 907-F67029, Strasbourg Cedex 1).

Example 20.7 Oxidation of desialylated EPO by galactose oxidase

In the presence of 16 μl catalase (6214 units/200ml) and 80 μl galactose oxidase (2250 units/ml, from Dactylium dendroides (Sigma-Aldrich, Steinheim, Germany), 4.485 mg of fully desialylated EPO was dissolved in 20 mM Incubate overnight at 37° C. in sodium phosphate buffer pH 6.8; add 20 μl of galactose oxidase in two portions 4 hours and 8 hours after the start of incubation.

Example 20.8 Preparation of EPO samples for biological detection

Purification of EPO from periodate- or galactose oxidase-oxidized EPO protein preparations incubated with activated HES

Purification of EPO samples (removal of unreacted HES derivatives) was performed at room temperature. EPO incubation mixture (approximately 5 mg EPO protein) was diluted 1:10 with buffer A (20 mM N-morpholine propanesulfonic acid [MOPS/NaOH] in double distilled water, pH 8.0) at a flow rate of 0.5 ml/min The sample was loaded onto a column containing 3 ml of Q-SepharoseHP (Pharmacia No. 17-1014-03, Lot No. 220211 ) equilibrated with 10 column volumes (CV) of buffer A. Wash the column with 6-8 column volumes of buffer A (flow rate = 0.8ml/min), use buffer B (20mM morpholineethanesulfonic acid [MES/NaOH, 0.5M NaCl, in double distilled water, pH6.5 ) was eluted at a flow rate of 0.5ml/min. EPO was detected by UV absorbance at 280 nm and eluted into about 6 ml. The column was regenerated with 3 column volumes of buffer C (20 mM MES, 1.5M NaCl in _H2O , adjusted to pH 6.5) and re-equilibrated with 10 column volumes of buffer A (flow rate = 0.7 ml/min ).

EPO eluates from the Q-Sepharose step were buffer exchanged with Vivaspin concentrators and phosphate-buffered saline (PBS), with 3 centrifugation cycles per sample; samples were adjusted to 2 ml with PBS and stored at -20°C.

Since the basal EPO form does not bind Q-Sepharose under the conditions employed and was found to be present in the flow-through along with unreacted HES derivative, only <25% partial sialylation was obtained from the Q-Sepharose eluate, Subsequent mild periodate oxidation of the HES-modified EPO form.

Example 20.9 High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

Purified native and desialylated oligonucleotides were analyzed by high pH anion exchange (HPAE) chromatography with pulsed amperometric detection (PAD) using a Dionex BioLC system (Dionex, USA) equipped with a CarboPac PA1 column (0.4 × 25 cm). Sugars (Schroter et al., 1999; Nimtz et al., 1999). The detector potential (E) and pulse duration (T) are: E1: +50mV, T1: 480ms; E2: +500mV, T2: 120ms; E3: -500mV, T3: 60ms, and the output range is 500-1500nA. The oligosaccharides were then injected onto a CarboPac PAl column equilibrated with 100% solvent A. For desialylated oligosaccharides, elution was performed using a linear gradient of solvent B (0-20%) in 40 minutes, followed by a linear increase of solvent B from 20% to 100% in 5 minutes (flow rate: 1 ml· min ^-1 ). Solvent A was 0.2M NaOH dissolved in double distilled water and solvent B consisted of 0.6M NaOAc dissolved in solvent A. For natural oligosaccharides, the column was equilibrated with 100% solvent C (0.1M NaOH in double-distilled water), followed by a linear gradient (0-35%) of solvent D over 48 minutes, followed by solvent D from 35 The % linearity increased to 100%, and elution was performed (flow rate: 1 ml·min ⁻¹ ). Solvent D consisted of 0.6M NaAc dissolved in solvent C.

Example 20.10 Monosaccharide composition analysis of N-glycans, HES-modified N-glycans and EPO proteins by GC-MS

Analysis of the corresponding methyl glycosides by GC/MS after methanolysis, N-reacetylation and trimethylsilylation [Chaplin, M.F. (1982) A rapid and sensitive method for the analysis of carbohydrates. Anal.Biochem.123 , 336-341] to analyze monosaccharides. These analyzes were performed with a Finnigan GCQ ion trap mass spectrometer (Finnigan MAT corp., San Jose, CA) operating in positive ion EI mode equipped with a 30 m DB5 capillary column. Temperature program: keep at 80°C for 2 minutes, then increase by 10°C per minute to 300°C.

Identify monosaccharides based on retention time and characteristic fragmentation patterns. The results of uncorrected electronic peak integrations were used for quantification. Monosaccharides that give rise to more than one peak due to anisomerization and/or the presence of furan and pyran forms are quantified by summing all major peaks. 0.5 μg of myo-inositol was used as an internal standard compound.

Example 20.11 Results

Example 20.11(a) Identification of N-glycans of weakly acid-treated (partially desialylated) EPO-GT-1

As shown in Figure 9, EPO-GT-1 preparations treated with mild acid for 5, 10 or 60 minutes were analyzed by SDS-PAGE before and after incubation with N-glycosidase to release N-linked oligosaccharides. HPAEC-PAD oligosaccharide mapping was performed on N-linked oligosaccharides (Figure 10). Untreated EPO-GT-1 contained >90% of N-linked oligosaccharides with 3 or 4 sialic acid residues, while <40% of carbohydrate chains contained 3 or 4 sialic acid residues after incubation for 5 minutes in the presence of mild acid 4 sialic acid residues. HPAEC-PAD of desialylated N-glycans showed that the proportion of neutral oligosaccharides detected in untreated EPO-GT-1 remained stable in preparations treated with acid for 5, 10 or 60 minutes. MALDI/TOF-MS of desialylated glycans showed that <90% proximal fucose was present after mild acid treatment of the protein.

Example 20.11(b) Identification of periodate-treated EPO-GT-1

The SDS-PAGE mobilities of mild periodate-treated EPO forms pre-acid-treated for 5 and 10 minutes or left untreated are compared in FIG. 12 . The conditions used for periodate oxidation of sialic acid did not alter the SDS-PAGE profile of the EPO preparation (compare Figure 9). Oxidation of sialic acid resulted in a shift of oligosaccharides towards earlier elution times in the HPAEC-PAD analysis (compare Figures 10 and 13).

Example 20.11(c) Identification of HES-modified EPO derivatives

(aa) HES modification of EPO-GT-1-A with HES derivative X produced according to Example 14.4 (time course)

400 μg of hydroxylamine-modified HES derivative X was added to 20 μg of EPO-GT-1-A (mild periodate oxidized EPO, untreated before mild periodate oxidation) dissolved in 20 μl of 0.5M NaOAc buffer pH 5.5. After acid hydrolysis), the reaction was stopped by freezing the samples in liquid nitrogen after 30 minutes, 2, 4, 17 hours, respectively. Samples were then stored at -20°C until further analysis.

Add SDS-PAGE sample buffer, heat the sample to 90°C, and load it on the SDS-gel. As shown in Figure 14, prolonged incubation times resulted in increased migration towards higher molecular weight proteins. After 17 hours of incubation in the presence of hydroxylamine-modified HES derivative X, a diffuse Coomassie blue-stained protein band was detected, which migrated in the region between 60-11 KDa according to the position of the molecular weight standard (see Figure 14 left part of ). After treatment with N-glycosidase, most proteins migrated to the position of de-N-glycosylated EPO (see Figure 14, right gel; arrow A indicates the migration position of N-glycosidase; arrow B indicates de-N-glycosidase -Position of glycosylated EPO; presumably the diffuse protein band seen in the region between 28KDa and 36KDa molecular weight standards represents the EPO type modified by HES at the O-glycosylation site of the molecule. Regarding the specificity of N-glycosidase , we conclude from this result that the HES-modification actually occurs at the periodate-oxidized sialic acid residues of the glycans of the EPO protein.

(bb) Identification of HES-EPO conjugates

HES-EPO conjugate I (derived from EPO-GT-1 after mild periodate oxidation, that is, derived from EPO-GT-1-A), II (derived from acid hydrolysis for 5 minutes and mild periodate Oxidized EPO-GT-1), III (EPO-GT-1 derived from acid hydrolysis for 10 min and mild periodate oxidation) were synthesized as previously described. A control incubation (K) was included under the same buffer conditions, which included unmodified EPO-GT-1 and added an equal amount of unmodified HES. These incubation mixtures were further purified for subsequent biochemical analysis of HES-EPO derivatives.

Incubations of HES-EPO conjugates I, II and III and control incubation K were subjected to a Q-Sepharose purification step as described in "Materials and Methods" (Example 20.8) to remove excess unreacted HES reagent, which Expected to occur in flow-through from ion exchange columns. Due to the abundance of the basal EPO form in pre-acid-treated samples II and III, we expected the flow-through to contain a considerable amount of modified EPO product from these incubations. As shown in Fig. 15, almost all EPO derived from sample I was retained by the Q-Sepharose column, while only about 20–30% in samples III and II were recovered in the fractions eluted with high salt concentration. In SDS-PAGE, all protein species produced by incubation with HES derivative X had higher apparent molecular weights in the flow-through and in the fractions eluted with high salt compared to the control EPO.

To characterize HES-modified EPO samples A and K in more detail (see Figure 13), we compared with the periodate oxidized form EPO-GT-1-A. Samples were subjected to N-glycosidase treatment, and as shown in Figures 16a and 16b, the release of N-glycans produced two low molecular weight bands at O-glycosylated and non-glycosylated EPO-type positions in standard EPO preparations. For sample A, another band was detected that migrated at the position of the 28 KDa molecular weight marker, suggesting HES-modification at the O-glycans of this EPO variant (see Example 20.11(c)(aa)). This band (as well as the highly HES-modified high molecular weight form of N-glycosylated EPO, see Figures 16a and 16b) disappeared after mild hydrolysis of the sample, which is consistent with the periodate oxidation of erythropoietin in saliva It is consistent with the view that HES modification occurs at acid residues.

An aliquot of the N-glycosidase-incubated mixture is hydrolyzed using conditions that allow complete removal of sialic acid residues (and sialic acid-linked HES derivatives) from the oligosaccharide; after neutralization, the mixture is adsorbed onto a small Hypercarb column for desalination. The column was washed extensively with water, followed by elution of bound neutral oligosaccharides with 40% acetonitrile in water containing 0.1% trifluoroacetic acid. The resulting oligosaccharides were subjected to MALDI/TOF-MS. The spectra of the desialylated oligosaccharide fractions from sample A, EPO-GT-1-A and sample K show the same mass of complex oligosaccharides: m/z = 1810 Da (two-armed), 2175 = three-armed, 2540 = four branches, 2906 = four branches + 1 N-acetyllactosamine repeat, 3271 = four branches + 2 N-acetyllactosamine repeats; detected corresponding to lack of fucose (-146) and galactose ( -162), which can be attributed to the acid hydrolysis conditions used to remove sialic acid (see MALDI - Figures 19, 20, 21).

In a parallel experiment, the N-glycosidase digestion mixture was adsorbed onto a 1 ml RP-C18 column (oligosaccharides had not been hydrolyzed with acid beforehand) and eluted with 5% acetonitrile in water containing 0.1% TFA; under these conditions, The EPO protein was completely retained on the RP-material and the oligosaccharides were washed off the column with 5% acetonitrile in water containing 0.1% TFA. The de-N-glycosylated EPO protein was eluted with 70% acetonitrile in water containing 0.1% TFA. Oligosaccharide fractions obtained after neutralization of N-glycosidase-treated sample A, EPOGT-1-A and sample K through the RP-C18 step were desalted with Hypercarb columns as previously described. HPAEC-PAD mapping was performed on isolated oligosaccharides before (see Figure 17) and after (see Figure 18) mild acid treatment under conditions capable of quantitative removal of sialic acid from glycans.

The HPAEC-PAD profile of the natural material obtained from the HES-modified sample A showed only negligible oligosaccharide signals, whereas the EPOGT-1-A-derived oligosaccharides showed the same pattern as the sample shown in Figure 13 (termed mild periodate Treated EPO-GT-1 sample) HPAEC-PAD profile of the same glycans. The elution profile of oligosaccharides obtained from the control EPO sample (K) yielded the expected pattern (comparative plot in Figure 10). For comparison, the native oligosaccharide profile of the international BRP-EPO standard for comparison and reference standard was included.

After weak acid hydrolysis, all oligosaccharide preparations showed the same elution profile of neutral oligosaccharide structures (see Figure 18) with qualitative and quantitative composition of bi-, tri-, and tetra-branched complex carbohydrate chains, as in The EPO preparation used as the starting material for this study is described in the Methods section. This result confirms that HES-modification of EPO samples leads to covalent attachment of HES derivatives, which can be detached from EPO-proteins by N-glycosidase, and can be desialylated from carbohydrates using mild acid treatment conditions. N-glycans are removed (see Figure 16a+b), so the derivative is acid labile.

(cc) Monosaccharide composition analysis of HES-EPO and HES-EPO N-glycans by GC-MS

To further confirm the HES-modification of EPO at molecular N-glycans, the EPO samples were digested with N-glycosidase, the EPO protein was adsorbed onto the RP-C18 column, while the oligosaccharide species were washed away as described above. As shown in Table 3, glucose and hydroxyethylated glucose derivatives were detected only in the EPO protein with HES modification at cysteine residues and in the oligosaccharide fraction of EPO sample A2.

Example 20.11(d) In vivo bioactivity detection of HES-modified EPO

EPO bioassays were performed in normal erythrocytic mouse lines according to the method described in the European Pharmacopoeia; the laboratory for EPO assays used the international BRP EPO reference standard product. For HES-modified EPO A2 preparations, the average specific activity measured was 294600 units per mg of EPO protein, indicating that the specific activity was about 3 times higher than that of the international BRP EPO reference standard preparation that was also tested for activity.

The results of the study are summarized in Table 4.

References for Examples 13-20:

Nimtz M, Noll G, Paques EP, Conradt HS.

Carbohydrate structures of a human tissue plasminogen activator expressed in recombinant Chinese hams ter ovary cells.

FEBS Lett.1990 Oct.1; 271(1-2): 14-8

Dorner AJ, Wasley LC, Kaufman RJ.

Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treatedChinese hamster ovary cells.

J Biol Chem.1989 Dec 5;264(34):20602-7

Mueller PP, Schlenke P, Nimtz M, Conradt HS, Hauser H

Recombinant glycoprotein quality inproliferation-controlled BHK-21cells.

Biotechnol Bioeng. 1999 Dec 5;65(5):529-36

Nimtz M, Martin W, Wray V, Kloppel KD, Augustin J, Conradt HS.

Structures of sialylated oligosaccharides of humanerythropoietine expressed in recobminant BHK-21 cells.

Eur J Biochem.1993 Apr.1;213(1):39-56

Hermentin P, Witzel R, Vliegenthart JF, Kamerling JP, Nimtz M. Conradt HS.

A strategy for the mapping of N-glycans by high-phanion-exchange chromatography with pulsed amperometric detection.

Anal Biochem.1992 Jun; 203 (2): 281-9

Schroter S, Derr P, Conradt HS, Nimtz M, Hale G, Kirchhoff C.

Male specific modification of human CD52.

J Biol Chem.1999 Oct.15;274(42):29862-73

Table 1 connector type Functional group 1: Reaction with peptides, especially EPO Functional group 2: Reaction with HES A Hydrazide (aldehyde reactive) Maleimide (SH reactive) B Hydrazide (aldehyde reactive) Pyridyl disulfide (SH reactive) C Iodoalkyl (SH reactive) N-Succinimidyl Ester (Amine Reactive) D. Bromoalkyl (SH reactive) N-Succinimidyl Ester (Amine Reactive) E. Maleimide (SH reactive) N-Succinimidyl Ester (Amine Reactive) f Pyridyl disulfide (SH reactive) N-Succinimidyl Ester (Amine Reactive) G Vinyl Sulfone (SH Reactive) N-Succinimidyl Ester (Amine Reactive)

Table 2

table 3

Monosaccharide composition analysis of glycans from HES-modified EPO and control samples **Monosaccharide I. Glycans from A2 II. Glycans from EPO-GT-1A III. Glycans from K2 III. Glycans from A2 IV. Glycans from EPO-GT-1A V. Glycans from K2 VI. Cysteine-modified EPO protein* Fucose 1935 3924 2602 2246 4461 2601 2181 Mannose 6028 11020 9198 6379 11668 6117 6260 Galactose 8886 19935 14427 10570 16911 11555 10386 glucose 17968 --- --- 21193 Trace Trace 33021 GlcNAc 7839 21310 14440 11360 15953 10503 10498 GlcHe1 5583 --- --- 5926 --- --- 14857 GlcHe2 1380 --- --- 1552 --- --- 3775 NeuNAc 5461 822 4504 3895 4871 13562 13003 Inositol 1230 2310 620 2050 1320 1134 1087 * Compositional analysis was performed on equal amounts of Cys-HES-modified EPO protein; EPO protein was separated from the HES-incubation mixture by chromatography on a Q-Sepharose column as described above and desalted by centrifugation using a Vivaspin 5 separation device. **Monosaccharide determination by single-shot GC of per-trimethylsilylated methyl glycosides; electron integration values for peaks are given, these values are not corrected for losses during derivatization and recovery of each compound .

Table 4 sample number sample discription Calculated specific activity of EPO samples (measured according to A280nm and RP-HPLC) 850247 1. HES-modified EPO A2 344000U/mg 850248 2. EPO-GT-1-A 82268U/mg 850249 3. Control EPO K2 121410U/mg 850250 4. BRP EPO standard 86702U/mg 850251 1. Dilute with 4 times the volume of PBS 309129U/mg 850252 2. Dilute with 4 times the volume of PBS 94500U/mg 850253 3. Dilute with 4 volumes of PBS 114100U/mg 850254 4. Dilute with 4 times the volume of PBS 81200U/mg 850255 1. Dilute with 4 times the volume of PBS 230720U/mg

Claims (85)

1. hydroxyalkyl starch (HAS)-erythropoietin (EPO) conjugate (HAS-EPO) that contains one or more HAS molecules, wherein each HAS passes through with EPO

A) carbohydrate part; Or

B) thioether coupling.

2. the HAS-EPO of claim 1, wherein EPO has the aminoacid sequence of people EPO.

3. claim 1 or 2 HAS-EPO, wherein EPO contains by N-and/or O-and connects one or more carbohydrate side chains that glycosylation is connected with EPO.

4. the HAS-EPO of claim 3, wherein Mammals particularly in the production process in people, insect or the yeast cell, described carbohydrate side chain is connected with EPO.

5. each HAS-EPO among the claim 1-4, wherein HAS is by linkers and EPO coupling.

6. each HAS-EPO among the claim 3-5, wherein HAS is by carbohydrate part and EPO coupling, and this carbohydrate partly is the part of carbohydrate side chain and preferably oxidized.

7. the HAS-EPO of claim 6, the wherein semi-lactosi of HAS and carbohydrate side chain or sialic acid residues coupling.

8. each HAS-EPO among the claim 1-7, wherein the S atom in the thioether derives from the halfcystine of naturally occurring halfcystine or interpolation.

9. the HAS-EPO of claim 8, wherein EPO has the aminoacid sequence of people EPO, and naturally occurring halfcystine is halfcystine 29 and/or 33.

10. the HAS-EPO of claim 9, wherein HAS and halfcystine 29 couplings, halfcystine 33 is replaced into another kind of amino acid.

11. the HAS-EPO of claim 9, wherein HAS and halfcystine 33 couplings, halfcystine 29 is replaced into another kind of amino acid.

12. each HAS-EPO among the claim 8-11 is wherein by being that halfcystine adds halfcystine with naturally occurring amino-acid substitution.

13. the HAS-EPO of claim 12, wherein EPO is people EPO, is Serine 126 by the metathetical amino-acid residue.

14. each HAS-EPO among the claim 1-13, wherein each EPO molecule has 1-12, preferred, 1-6 or 1-3,1-4 HAS molecule most preferably.

15. each HAS-EPO among the claim 1-14, wherein HAS is selected from hydroxyethylamyle, hydroxypropylated starch and hydroxyl butyl starch.

16. the HAS-EPO of claim 15, wherein HAS is hydroxyethylamyle (HES).

17. the HAS-EPO of claim 16, wherein the molecular weight of HES is 1-300kDa, preferred 5-100kDa.

18. the HAS-EPO of claim 16 or 17, wherein the HES demonstration replaces ratio with respect to molar substitution and the C2 of 2-20: the C6 of the 0.1-0.8 of hydroxyethyl.

19. a method of producing hydroxyalkyl starch (HAS)-erythropoietin (EPO) conjugate (HAS-EPO) comprises the following steps:

A) provide the EPO that can react with the HAS of modification,

B) provide can with the HAS of the modification of EPO reaction in the step a) and

C) make the EPO of step a) and the HAS reaction of step b), thereby produce the HAS-EPO that contains one or more HAS molecules, wherein each HAS passes through with EPO

I) carbohydrate part; Or

Ii) thioether coupling.

20. the method for claim 19, wherein EPO has the aminoacid sequence of people EPO.

21. the method for claim 19 or 20, wherein EPO is that reorganization produces.

22. each method among the claim 19-21, wherein EPO contains by N-and/or O-and connects one or more carbohydrate side chains that glycosylation is connected with EPO.

23. the method for claim 22, wherein Mammals particularly in the production process in people, insect or the yeast cell, described carbohydrate side chain is connected with EPO.

24. the method for claim 22 or 23, wherein HAS passes through carbohydrate part and EPO coupling, and this carbohydrate partly is the part of carbohydrate side chain.

25. the method for claim 24, wherein in step a), at least one carbohydrate part of the one or more carbohydrate side chains by oxidation EPO, preferably at least one terminal sugar unit, more preferably semi-lactosi is modified EPO.

26. the method for claim 25 is wherein removed the terminal sugar unit of terminal sialic acid rear oxidation at partially or completely (enzyme and/or chemistry).

27. the method for claim 25 or 26, wherein in step c), the terminal sugar unit coupling of the HAS of modification and oxidation.

28. each method among the claim 19-27, wherein EPO contains at least one free SH base.

29. the method for claim 28, wherein free SH base is the part of the halfcystine of naturally occurring halfcystine or interpolation.

30. the method for claim 29, wherein EPO has the aminoacid sequence of people EPO, and naturally occurring halfcystine is halfcystine 29 and/or 33.

31. the method for claim 30, wherein halfcystine 33 is replaced into another kind of amino acid, HAS that modifies in step c) and halfcystine 29 couplings.

32. the method for claim 30, wherein halfcystine 29 is replaced into another kind of amino acid, HAS that modifies in step c) and halfcystine 33 couplings.

33. each method among the claim 29-32 is wherein by being that halfcystine adds halfcystine with naturally occurring amino-acid substitution.

34. the method for claim 33, wherein EPO is people EPO, is Serine 126 by the metathetical amino-acid residue.

35. the method for claim 33 or 34, wherein in step c), the halfcystine coupling of the HAS of modification and interpolation.

36. each method among the claim 19-35, wherein modify HAS, if the carbohydrate part coupling of feasible HAS and oxidation, then it contains free hydrazides, azanol, mercaptan or Urea,amino-functional group, if perhaps HAS and SH base coupling, then it contains free maleimide, disulfide or Haloacetamide functional group.

37. each method among the claim 19-36, wherein step c) is containing 10% weight H at least ₂Carry out in the reaction medium of O.

38. each method among the claim 19-37, wherein HAS is by linkers and EPO coupling.

39. each method among the claim 19-38, wherein HAS is hydroxyethylamyle, hydroxypropylated starch or hydroxyl butyl starch, preferred hydroxyethylamyle (HES).

40. the method for claim 39, wherein HES has the character as each limited in claim 17 or 18.

41. HAS-EPO by each method acquisition among the claim 19-40.

42. the HAS-EPO of claim 41, it has the feature that each limited among the claim 1-18.

43. be used for existing of human or animal body methods of treatment according to claim 1-18,41 or 42 each HAS-EPO.

44. a pharmaceutical composition, it contains among the with good grounds claim 1-18,41 or 42 each HAS-EPO.

45. the pharmaceutical composition of claim 44 further contains at least a pharmaceutically acceptable carrier.

46. according to each the application of HAS-EPO in the medicine of preparation treatment anaemia disease or hematopoietic disorder disease among the claim 1-18,41 or 42.

47. contain hydroxyalkyl starch (the HAS)-polypeptide conjugate (HAS-polypeptide) of one or more HAS molecules, wherein each HAS passes through with polypeptide

A) carbohydrate part; Or

B) thioether coupling.

48. the HAS-polypeptide of claim 47, wherein polypeptide is the humanized.

49. the HAS-polypeptide of claim 47 or 48, wherein polypeptide is selected from erythropoietin, interleukin particularly interleukin-2, IFN-β, IFN-α, CSF, interleukin 6 and therapeutic antibodies.

50. each HAS-polypeptide among the claim 47-49, wherein polypeptide contains by N-and/or O-and connects one or more carbohydrate side chains that glycosylation is connected with polypeptide.

51. the HAS-polypeptide of claim 50, wherein Mammals particularly in the production process in people, insect or the yeast cell, described carbohydrate side chain is connected with polypeptide.

52. each HAS-polypeptide among the claim 47-51, wherein HAS is by linkers and polypeptide coupling.

53. each HAS-polypeptide among the claim 49-52, wherein HAS is by carbohydrate part and polypeptide coupling, and this carbohydrate partly is the part of carbohydrate side chain, and preferably oxidized.

54. the HAS-polypeptide of claim 53, wherein the galactose residue coupling of HAS and carbohydrate side chain.

55. each HAS-polypeptide among the claim 47-54, wherein the S atom in the thioether derives from the halfcystine of naturally occurring halfcystine or interpolation.

56. the HAS-polypeptide of claim 55 is wherein by being that halfcystine adds halfcystine with naturally occurring amino-acid substitution.

57. each HAS-polypeptide among the claim 47-56, wherein each peptide molecule has 1-12, preferred 1-6 or 1-3,1-4 HAS molecule most preferably.

58. each HAS-polypeptide among the claim 47-57, wherein HAS is selected from hydroxyethylamyle, hydroxypropylated starch and hydroxyl butyl starch.

59. the HAS-polypeptide of claim 58, wherein HAS is hydroxyethylamyle (HES).

60. the HAS-polypeptide of claim 59, wherein the molecular weight of HES is 1-300kDa, preferred 5-100kDa.

61. each HAS-polypeptide in claim 59 or 60, wherein the HES demonstration is with respect to the mole replacement degree of the 0.1-0.8 of hydroxyethyl and the C of 2-20 ₂: C ₆Replace ratio.

62. a method of producing hydroxyalkyl starch (HAS)-polypeptide conjugate (HAS-polypeptide) comprises the following steps:

A) provide the polypeptide that can react with the HAS of modification,

B) provide can with the HAS of the modification of polypeptide reaction in the step a) and

C) make the polypeptide of step a) and the HAS reaction of step b), thereby produce the HAS-polypeptide that contains one or more HAS molecules, wherein each HAS passes through with polypeptide

I) carbohydrate part; Or

Ii) thioether coupling.

63. the method for claim 62, wherein polypeptide is the humanized.

64. the method for claim 62 or 63, wherein polypeptide is selected from erythropoietin, interleukin particularly interleukin-2, IFN-β, IFN-α, CSF, interleukin 6 and therapeutic antibodies.

65. each method among the claim 62-64, wherein polypeptide is that reorganization produces.

66. each method among the claim 62-65, wherein polypeptide contains by N-and/or O-and connects one or more carbohydrate side chains that glycosylation is connected with polypeptide.

67. the method for claim 66, wherein Mammals particularly carbohydrate side chain described in the production process in people, insect or the yeast cell be connected with polypeptide.

68. the method for claim 66 or 67, wherein HAS is by carbohydrate part and polypeptide coupling, and this carbohydrate partly is the part of carbohydrate side chain.

69. the method for claim 68, wherein in step a), at least one carbohydrate part of the one or more carbohydrate side chains by the oxidation polypeptide, preferably at least one terminal sugar unit, more preferably semi-lactosi is modified this polypeptide.

70. the method for claim 69 is wherein removed the terminal sugar unit of terminal sialic acid rear oxidation at partially or completely (enzyme and/or chemistry).

71. the method for claim 69 or 70, wherein in step c), the terminal sugar unit coupling of the HAS of modification and oxidation.

72. each method among the claim 62-71, wherein polypeptide contains at least one free SH base.

73. the method for claim 72, wherein free SH base is the part of the halfcystine of naturally occurring halfcystine or interpolation.

74. each method among the claim 62-73 is wherein by being that halfcystine adds halfcystine with naturally occurring amino-acid substitution.

75. each method in claim 73 or 74, wherein in step c), the halfcystine coupling of the HAS of modification and interpolation.

76. each method among the claim 62-75, wherein modify HAS, if make HAS and oxidation the coupling of carbohydrate part contain free hydrazides, azanol, mercaptan or Urea,amino-functional group, if perhaps HAS and the basic coupling of SH then contain free maleimide, disulfide or Haloacetamide functional group.

77. each method among the claim 62-76, wherein step c) is containing 10% weight H at least ₂Carry out in the reaction medium of O.

78. each method among the claim 62-78, wherein HAS is by linkers and polypeptide coupling.

79. each method among the claim 62-78, wherein HAS is hydroxyethylamyle, hydroxypropylated starch or hydroxyl butyl starch, preferred hydroxyethylamyle (HES).

80. the method for claim 79, wherein HES has the character as each limited in claim 60 or 61.

81. can be by the HAS-polypeptide of each method acquisition among the claim 62-80.

82. the HAS-polypeptide of claim 41, it has the feature that each limited among the claim 47-61.

83. in the human or animal body methods of treatment, use according to each HAS-polypeptide among the claim 47-61,81 or 82.

84. a pharmaceutical composition, it contains among the with good grounds claim 47-61,81 or 82 each HAS-polypeptide.

85. the pharmaceutical composition of claim 84 further contains at least a pharmaceutically acceptable carrier.

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